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MicroRNAs regulate osteogenesis and chondrogenesis
Abstract
MicroRNAs (miRNAs) are a class of small molecules and non-coding single strand RNAs that regulate gene expression at the post-transcriptional level by binding to specific sequences within target genes. miRNAs have been recognized as important regulatory factors in organism development and disease expression. Some miRNAs regulate the proliferation and differentiation of osteoblasts, osteoclasts and chondrocytes, eventually influencing metabolism and bone formation. miRNAs are expected to provide potential gene therapy targets for the clinical treatment of metabolic bone diseases and bone injuries. Here, we review the recent research progress on the regulation of miRNAs in bone biology, with a particular focus on the miRNA-mediated control mechanisms of bone and cartilage formation.
... After being confirmed in the human body Zhu et al., 2011), microRNAs have attracted great attention for clinical applications because of their great impact on metabolism (Luo et al., 2011), an example of critical cellular activities in the body. Other critical cellular activities include programmed cell death (Dong, Yang, Guo, & Kang, 2012) and differentiation and proliferation (Dong et al., 2012;Sharon et al., 2019) (Fig. 3.6). ...
... After being confirmed in the human body Zhu et al., 2011), microRNAs have attracted great attention for clinical applications because of their great impact on metabolism (Luo et al., 2011), an example of critical cellular activities in the body. Other critical cellular activities include programmed cell death (Dong, Yang, Guo, & Kang, 2012) and differentiation and proliferation (Dong et al., 2012;Sharon et al., 2019) (Fig. 3.6). ...
Polymeric nanoparticles (PNPs) are tiny solid and colloidal particles with sizes ranging from a few nanometers to 1000 nm, with versatile structures and morphologies. It is an innovative form of materials derived through nanotechnology for technological improvement in drug delivery and other biomedical applications. PNPs are manufactured through the use of both natural and synthetic polymers using different techniques for desired properties. Research has shown the different stimulus responses of these manufactured PNPs. This article reviews the different possible types of PNPs, up-to-date improvements in the synthesis of different PNPs, and the different biomedical applications of PNPs. There are more discoveries to be unleashed year after year, especially with regard to the most recent medical issues such as viral diseases and other illnesses.
... The expressions of miRs were detected by Tq-PCR. In accordance with the previous reports [24,[40][41][42][43][44][45][46][47][48][49][50][51], miR-675, miR125a, miR-199b were upregulated by AdH19, and miR-132, miR-27b, miR-34a, miR-449a, miR-449b, miR-93, miR-106b, miR-107, miR-129-1, miR-17 and miR-let-7d were downregulated by AdH19 at different levels ( Figure 3). These results indicate that except working as pre-miR of miR-675, AdH19 can also working as ceRNA to competitively combine with some miRs. ...
... In the present study, we also explored whether adenovirus mediated overexpression of H19 acting as ceRNA. Among the 14 miRs which we selected may associated with H19 [24,[40][41][42][43][44][45][46][47][48][49][50][51], we found that 3 miRs (miR-675, miR-125, miR-199b) were upregulated by overexpression of H19, and 11 miRs (miR-132, miR-27b, miR34a, miR-449a, miR-449b, miR93, miR-106b, miR-107, miR-129-1, miR-17 and miR-let-7d) were downregulated by overexpression of H19. Be consistent with the previous reports, H19 was found to be act as ceRNA or molecular sponge of miR-107 [21,43], miR-let-7 [24,44,45], miR-93 [68], miR-106b [69], miR-17 [51]. ...
Background:
We previously constructed AdEasy system for rapid generation of recombinant adenovirus expressing coding genes. However, it is unclear if AdEasy system could be used for exogenously expression of long noncoding RNAs (lncRNAs). Here we investigated how to overexpress lncRNA H19 with AdEasy system and identified the effect of overexpression H19 on mesenchymal stem cells (MSCs) osteogenic differentiation.
Methods:
H19 fragment 1 and H19 fragment 2 were amplified from mouse genomic DNA separately and then connected for full-length H19. H19 was firstly subcloned to homemade pMOK plasmid, then H19 was cut off from pMOK-H19 and subcloned to recombinant adenovirus plasmid. After homologous recombination in AdEasier cells (BJ5183 cell), packing in mammalian packaging cell line and amplification in 293pTP cells, high titer AdH19 was obtained. Immortalized mouse adipose-derived progenitors (iMADs) were infected by AdH19 with different infection rate, the expression of H19, H19 related microRNAs (miRs) and osteogenic differentiation markers were determined by TqPCR. Alkaline phosphatase (ALP) activities and matrix mineralization were determined by ALP assays and Alizarin red S staining respectively.
Results:
AdEasy system was suitable for rapid generation and production of H19, AdH19 can effectively overexpress H19 and serve as functional lncRNA in mesenchymal stem cells (MSCs). Higher dosage of AdH19 inhibited osteogenic differentiation of MSCs, however, lower dosage of AdH19 promoted osteogenic differentiation of MSCs.
Conclusions:
We firstly reported the method for the generation of functional lncRNA with AdEasy system, and identified the biphasic effect of H19 on MSCs osteogenic differentiation.
... There are many reports on critical roles miRNAs play in biology, such as differentiation, development, proliferation, and tumorigenesis. Moreover, several miRNAs have been identified to modulate cartilage differentiation and degradation [6][7][8]. For example, miR-365 targets histone deacetylase 4 and enhances chondrogenesis [9], and miR-145 inhibits early chondrogenesis by targeting Sox9 [10]. ...
... Expression of 6 miRNAs implicated in chondrogenesis was evaluated by RT-qPCR at 0, 7, 14, 21 and 28 days of chondrogenic differentiation [6][7][8][9][10]. Our results showed that expression of chondrogenesis-related miRNAs miR-140, miR-199a, miR-365, and miR-675 increased gradually during the entire chondrogenesis process, whereas miR-145 levels gradually decreased. ...
Background
MicroRNAs (miRNAs) reportedly participate in the mesenchymal stem cell (MSC) chondrogenic differentiation regulation. We objected to examine how miR-218 regulate chondrogenic differentiation of synovium-derived MSCs (SDSCs) and the maturation of RCJ3.1C5.1 chondrocytes. SDSCs were sourced from the knee joint synovium of New Zealand white rabbits, and their multilineage differentiation potentials were examined. The level of miR-218 was measured during SDSC chondrogenic differentiation, together with determination of SDSCs chondrogenic markers and RCJ3.1C5.1 chondrocytes maturation markers expression level after transfection of miR-218 mimics/inhibitor.
Results
miR-218 expression was notably upregulated in early chondrogenesis but mostly ceased during the maturation phases of chondrogenic differentiation in SDSCs. The transfection of miR-218 mimics notably enhanced SDSCs chondrocytes differentiation, as evidenced by augmented expressions of chondrogenic markers (SOX9, COL2A1, ACAN, GAG, and COMP) in terms of mRNA and protein level, and the inhibition of miR-218 yielded opposite resutls. Additionally, miR-218 overexpression substantially suppressed the expression of osteogenic markers (ALP, BSP, COL1A1, OCN and OPN) during the early stage of chondrogenesis while increasing that of chondrogenic markers (SOX9, COL2A1, ACAN, GAG and COMP). However, miR-218 mimics notably suppressed maturation markers (CMP, COL10A1, MMP-13 and VEGF) expression in RCJ3.1C5.18 chondrocytes, and the miR-218 inhibitor promoted the expression of these maturation markers. We proposed miR-218 plays a regulatory role on 15-hydroxyprostaglandin dehydrogenase (HPGD), which plays a key role in chondrogenic differentiation, and this finding indicates that miR-218 directly regulates HPGD expression in SDSCs.
Conclusion
Our study suggests that miR-218 contributes to early chondrogenesis while suppressing later chondrocyte maturation. The miR-218-HPGD pathway offers us a perspective into how SDSCs differentiate into chondrogenic cells.
... Accumulating evidence has revealed that microRNA (miRNA)-mediated posttranscriptional regulation may coordinate transcription factors to determine cell fate [24]. miRNAs have been reported to regulate the diferentiation of osteogenic progenitor cells and the process of bone formation [25][26][27]. However, it is still unknown whether miRNAs also contribute to PEMF-promoted osteogenic diferentiation. ...
Background. Insufficient bone formation is the key reason for the imbalance of bone metabolism and one of the main mechanisms for the occurrence and deterioration of postmenopausal osteoporosis (PMOP). Accumulating evidence has demonstrated that pulsed electromagnetic field (PEMF), as a physiotherapy, can treat osteoporosis by promoting osteogenic differentiation in osteoblasts. However, little is known about its mechanisms. Methods. In vivo, ovariectomized mice were administered PEMF for 4 weeks, and skeletal analysis was conducted. In vitro, hydrogen peroxide-treated mouse osteoblast precursor cells with or without PEMF intervention were subjected to osteogenic differentiation testing and miRNA microarrays. The potential target miRNAs were validated, followed by gene expression assays to further clarify their regulatory relationships with target pathways. Results. We found that PEMF reduced bone loss in ovariectomized mice and promoted osteogenic differentiation of hydrogen peroxide-treated osteoblast precursor cells via downregulation of miR-6976-5p. Mechanistically, reduced miR-6976-5p enhanced the nuclear transport of phosphorylated Smad1/5/9 by upregulating Smad4, thereby activating the BMP/Smad pathway. Additionally, the administration of miR-6976-5p inhibitors successfully promoted osteogenic differentiation in vitro, and its antagomirs protected bone mass in vivo. miR-6976-5p mimics and agomirs acted in the opposite way. Conclusion. These results provide evidence that PEMF alleviates estrogen deficiency-induced bone loss by activating osteoblastic progenitor cells and maintaining their osteogenic differentiation and shed light on the mechanisms involved, which may provide a potential option for the clinical application of PEMF in PMOP.
... 203,212e214 H19 in endochondral ossification and angiogenesis H19 was identified to regulate chondrogenic differentiation and maintain the anabolic and catabolic activities of chondrocytes and endochondral ossification. 60,92,215 In mouse limbs, we detected that the expression level of H19 is high in the proliferation zone and decreases gradually from the prehypertrophic zone to the hypertrophic zone. In addition, we characterized that H19 is essential for chondrogenic differentiation and maintaining the proliferation of chondrocytes, and down-regulation of H19 promoted hypertrophic differentiation of chondrocytes and followed endochondral ossification. ...
Recent advances in deep sequencing technologies have revealed that, while less than 2% of the human genome is transcribed into mRNA for protein synthesis, over 80% of the genome is transcribed, leading to the production of large amounts of noncoding RNAs (ncRNAs). It has been shown that ncRNAs, especially long non-coding RNAs (lncRNAs), may play crucial regulatory roles in gene expression. As one of the first isolated and reported lncRNAs, H19 has gained much attention due to its essential roles in regulating many physiological and/or pathological processes including embryogenesis, development, tumorigenesis, osteogenesis, and metabolism. Mechanistically, H19 mediates diverse regulatory functions by serving as competing endogenous RNAs (CeRNAs), Igf2/H19 imprinted tandem gene, modular scaffold, cooperating with H19 antisense, and acting directly with other mRNAs or lncRNAs. Here, we summarized the current understanding of H19 in embryogenesis and development, cancer development and progression, mesenchymal stem cell lineage-specific differentiation, and metabolic diseases. We discussed the potential regulatory mechanisms underlying H19's functions in those processes although more in-depth studies are warranted to delineate the exact molecular, cellular, epigenetic, and genomic regulatory mechanisms underlying the physiological and pathological roles of H19. Ultimately, these lines of investigation may lead to the development of novel therapeutics for human diseases by exploiting H19 functions.
... MicroRNAs are small RNAs that regulate gene expression [164]. They give distinct cellular responses in both bone formation [165] and bone disease [163], which indicates the possibility of a diagnosis and prognosis for bone diseases and susceptibility to fracture [162]. ...
Infancy and adolescence are crucial periods for bone health, since they are characterized by intense physical growth and bone development. The unsatisfactory acquisition of bone mass in this phase has consequences in adult life and increases the risk of developing bone diseases at more advanced ages. Nutrient deficiencies, especially calcium and vitamin D, associated with a sedentary lifestyle; lack of sun exposure; and epigenetic aspects represent some of the main risk factors for poor bone quality. In addition, recent studies relate childhood obesity to impaired bone health; however, studies on the adiposity effects on bone health are scarce and inconclusive. Another gap concerns the implications of obesity on child sexual maturity, which can jeopardize their genetic potential bone mass and increase fracture risk. Therefore, we reviewed the analyzed factors related to bone health and their association with obesity and metabolic syndrome in adolescents. We concluded that obesity (specifically, accumulated visceral fat) harms bones in the infant–juvenile phase, thereby increasing osteopenia/osteoporosis in adults and the elderly. Thus, it becomes evident that forming and maintaining healthy eating habits is necessary during infancy and adolescence to reduce the risk of fractures caused by bone-metabolic diseases in adulthood and to promote healthy ageing.
... miRNAs bind to target messenger RNA (mRNA), leading to silencing of the target mRNA/gene expression through translational repression or degradation of mRNA [3]. Most miRNAs are preserved in animal species, indicating the evolutionary significance of these molecules as regulators of vital cellular activities, such as proliferation, differentiation, apoptosis, and metabolism [4][5][6][7]. Aberrant expression of miRNAs and/or disruption of their function play important roles in the development of various pathological conditions, including cancer [8]. The dysregulation of miRNA expression leads to improper control of the functions of a wide number of proteins and biological mechanisms impacting cancer-associated signaling processes [9,10]. ...
MicroRNAs (miRNAs) are non-protein-coding RNA molecules 20–25 nucleotides in length that can suppress the expression of genes involved in numerous physiological processes in cells. Accumulating evidence has shown that dysregulation of miRNA expression is related to the pathogenesis of various human diseases and cancers. Thus, strategies involving either restoring the expression of tumor suppressor miRNAs or inhibiting overexpressed oncogenic miRNAs hold potential for targeted cancer therapies. However, delivery of miRNAs to tumor tissues is a challenging task. Recent advances in nanotechnology have enabled successful tumor-targeted delivery of miRNA therapeutics through newly designed nanoparticle-based carrier systems. As a result, miRNA therapeutics have entered human clinical trials with promising results, and they are expected to accelerate the transition of miRNAs from the bench to the bedside in the next decade. Here, we present recent perspectives and the newest developments, describing several engineered natural and synthetic novel miRNA nanocarrier formulations and their key in vivo applications and clinical trials.
... MiRNAs are endogenous RNAs composed of a limited number of nucleotides that play a role in gene regulation at the post-transcriptional level [2]. After the action of miRNAs in humans has been demonstrated, much attention has been focused on the therapeutic applications of these powerful agents, due to their enormous influence on critical cellular processes such as proliferation, differentiation [3], metabolism [4] and programmed cell death [5]. There is growing evidence that miRNAs are involved in many cancers at different levels. ...
Aim: MiRNA's-143 and -206 are powerful apoptotic regulators in cancer cells. This study aimed to use miRNA-143- and 206-loaded poly(lactic-co-glycolic) acid (PLGA) nanoparticles conjugated with folic acid to induce apoptosis in the EL4 cancer cells. Materials & methods: The therapy was conducted in six groups: Treatment with both miRNAs simultaneously (mixed miRNAs), miRNA-206 treatment, miRNA-143 treatment, blank PLGA, blank polyethylenimine (PEI) and complex PEI-miRNAs. Results: In terms of viability, in mixed miRNAs, no synergistic effect was observed on EL4 cell elimination. However, in the single miRNA-206 group, a stronger apoptotic effect was observed than the mixed miRNAs group and single miRNA-143 group alone. Conclusion: MiRNAs' apoptotic induction effects in cancer cells were found to be remarkable.
... microRNAs (miRs) play a critical role in regulating cell proliferation and differentiation, acting as post-transcriptional regulatory factors. Some miRNAs can shape metabolism and bone formation, controlling the proliferation and differentiation of osteoblasts, osteoclasts, and chondrocytes [116]. miR-320 was recently discovered as a negative regulator of osteoblast differentiation of hMSCs [117]. ...
Cytokines and their receptors have a vital function in regulating various processes such as immune function, inflammation, haematopoiesis, cell growth and differentiation. The interaction between a cytokine and its specific receptor triggers intracellular signalling cascades that lead to altered gene expression in the target cell and consequent changes in its proliferation, differentiation, or activation. In this review, we highlight the role of the soluble type I cytokine receptor CRLF1 (cytokine receptor-like factor-1) and the Interleukin (IL)-6 cytokine CLCF1 (cardiotrophin-like cytokine factor 1) during development in physiological and pathological conditions with particular emphasis on Crisponi/cold-induced sweating syndrome (CS/CISS) and discuss new insights, challenges and possibilities arising from recent studies.
... MiRNAs play an important role in the pathogenesis of a variety of human diseases [23]. Some miRNAs have been shown to be involved in cartilage differentiation and degradation [7,8]. MiR-499 modulated CUGBP2 and MYBO at the post transcription level in stem cell proliferation and differentiation [23]. ...
Background
Chondrogenic differentiation of human adipose-derived stem cells (hADSCs) is important for cartilage generation and degradation. LncRNAs play an essential role in stem cell differentiation. However, the role and mechanism of lncRNA in hADSCs remain unclear. Our previous study showed that miR-490-5p was downregulated during chondrogenic differentiation of hADSCs. In this study, we investigated the effect and mechanism of lncRNA NONHSAT030515 interacting with miR-490-5p on chondrogenic differentiation of hADSCs.
Methods
Alcian blue staining was used to assess the deposition of chondromatrix proteins following chondrogenic differentiation of human adipose stem cells. Immunohistochemistry was used to evaluate the expression of collagenII. TargetScan, miRTarBase and miRDB database analyses were applied to find the miRNA and target genes of lncRNA NONHSAT030515. A dual luciferase experiment was conducted to identify the direct target of NONHSAT030515. pcDNA3.1- NONHSAT030515 transfection and sh- NONHSAT030515 treatment were conducted to verify the role of lncRNA NONHSAT030515 in chondrogenic differentiation. The levels of Aggrecan, SOX9 and COL2A1 were analyzed by qRT-PCR and Western blot assay.
Results
Alcian blue staining, immunocytochemical, qRT-PCR, and Western blot have determined that lncRNA NONHSAT030515 can promote the chondrogenic differentiation of hADSCs. MiR-490- 5p was the direct target of NONHSAT030515, while BMPR2 was the target gene. This result was confirmed by luciferase reporter assay. Up-regulation of NONHSAT030515 promoted BMPR2 protein expression and promoted chondrogenic differentiation, whereas down-regulation of NONHSAT030515 caused completely opposite results.
Conclusion
LncRNA NONHSAT030515 promotes the chondrogenic differentiation of hADSCs through increasing BMPR2 expression by regulating miR-490- 5p.
... Numerous studies suggest that microRNAs (miRs) play an important role in the differentiation of stem cells by suppressing their target gene expression at the posttranscriptional level (Dong et al. 2012). In this study, the data demonstrated miR-320 directly bound to the 3'UTR of the CRLF1. ...
Background
Osteoarthritis (OA) is the most prevalent chronic joint disease, and is hard to be cured at present. Cytokine receptor-like factor 1 (CRLF1) has been identified as an upregulated gene in OA cartilage. However, the precise identities and functions of CRLF1 in OA progression have remained to be fully elucidated.
Methods
We used a murine model of injury-induced OA (destabilization of medial meniscus, DMM) and BMSCs to investigate the specific biological functions and mechanisms of CRLF1.
Results
We found that CRLF1 was significantly increased in the DMM surgery-induced OA model and was down-regulated during chondrogenic differentiation of BMSCs. Luciferase reporter assays showed that CRLF1 was a direct target of miR-320 in BMSCs. miR-320 can reverse the effect of CRLF1 on cell proliferation, apoptosis and chondrogenic differentiation of BMSCs. Furthermore, knockdown of CRLF1 or over-expression of miR-320 can inhibit the apoptosis of primary chondrocytes.
Conclusion
Suppression of CRLF1 promotes the chondrogenic differentiation of BMSCs and protects cartilage tissue from damage in osteoarthritis via activation of miR-320.
... Bone repair in trauma, congenital malformations, infections, surgery, and radiotherapy largely depends on the capability of bone formation (Majidinia et al., 2018;Wang T. et al., 2020). Bone formation and resorption are affected by the balance between osteoblast and osteoclast activities, and osteoblast differentiation is a key stage in bone formation that is closely regulated by factors, such as microRNAs (miRNAs) (Dong et al., 2012;Chen et al., 2015;Castano et al., 2020). MiRNAs, which are small non-coding RNA molecules that participate in gene regulation (Ambros, 2004;Bartel, 2004), represent a potential class of bone repair therapeutics because they can enhance the capacity of osteoblast differentiation (Behera and Tyagi, 2018;Chen et al., 2019;Yang et al., 2019;Castano et al., 2020;Won Lee et al., 2020). ...
MicroRNAs let-7c and let-7f, two members of the let-7 family, were involved in regulating osteoblast differentiation and have an important role in bone formation. Let-7e-5p, which also belonged to the let-7 family, presented in the differentiation of adipose-derived stem cells and mouse embryonic stem cells. However, the role of let-7e-5p in osteoblast differentiation was unclear. Thus, this study aimed to elucidate the function of let-7e-5p in osteoblast differentiation and its mechanism. Firstly, we found that the let-7e-5p mimic promoted osteoblast differentiation but not the proliferation of MC3T3-E1 cells by positively regulating the expression levels of osteogenic-associated genes ( RUNX2 , OCN , OPN , and OSX ), the activity of ALP, and formation of mineralized nodules. Moreover, we ascertained that the let-7e-5p mimic downregulated the post-transcriptional expression of SOCS1 by specifically binding to the 3′ untranslated region of SOCS1 mRNA. Also, let-7e-5p-induced SOCS1 downregulation increased the protein levels of p-STAT5 and IGF-1, which were both modulated by SOCS1 molecules. Furthermore, let-7e-5p abrogated the inhibition of osteogenic differentiation mediated by SOCS1 overexpression. Therefore, these results suggested that let-7e-5p regulated the differentiation of MC3T3-E1 cells through the JAK2/STAT5 pathway to upregulate IGF-1 gene expression by inhibiting SOCS1. These findings may provide a new insight into the regulatory role of let-7e-5p in osteogenic differentiation and imply the existence of a novel mechanism underlying let-7e-5p-mediated osteogenic differentiation.
... MiRNAs are small, non-coding single-stranded RNAs speci c for tissues or developmental stages and play regulatory roles by binding to speci c sequences of target genes post transcription. Some miRNAs have been shown to be involved in cartilage differentiation and degradation [7][8]. Our previous study showed that miR-490-5p was down-regulated during chondrogenic differentiation [9]. ...
Aim: Chondrogenic differentiation of human adipose-derived stem cells (hADSCs) is an important tissue in cartilage generation and degradation. LncRNAs play an essential role in stem cell differentiation. However, the role and mechanism of lncRNA in hADSCs remain unclear. Our previous study showed that miR-490-5p was downregulated during chondrogenic differentiation of human adipose-derived stem cells (hADSCs). In this study, we investigated the effect and mechanism of lncRNA NONHSAT030515 interacting with miR-490-5p on chondrogenic differentiation of hADSCs.
Methods: Alcin blue staining was used to assess the deposition of chondromatrix proteins following chondrogenic differentiation of human adipose stem cells. Immunohistochemistry was used to evaluate the expression of collagenⅡ. TargetScan, miRTarBase and miRDB database analysis were applied to find the miRNA and target genes of lncRNA NONHSAT030515. A dual luciferase experiment was conducted to identify the direct target of NONHSAT030515. pcDNA3.1- NONHSAT030515 transfection and sh- NONHSAT030515 treatment were conducted to verify the role of lncRNA NONHSAT030515 in chondrogenic differentiation. The levels of Aggrecan, SOX9 and COL2A1 were analyzed by qRT-PCR and western blot assay.
Results: Alcian blue staining, immunocytochemical, qRT-PCR, and Western blot have determined that lncRNA NONHSAT030515 can promote the chondrogenic differentiation of hADSCs. By luciferase reporter assay, it was confirmed that miR-490- 5p was the direct target and the BMPR2 was the target gene of NONHSAT030515. Up-regulation of NONHSAT030515 promoted BMPR2 protein expression and promoted chondrogenic differentiation whereas down-regulation of NONHSAT030515 caused completely opposite results.
Conclusion: LncRNA NONHSAT030515 regulated by miR-490-5p promotes the chondrogenic differentiation of hADSCs targeting BMPR2.
... However, it has been reported that PPARG gene expression, a marker of adipocyte, is regulated by hsa-miR-192 induction (72). The inhibition or induction of hsa-miR-9 and hsa-miR-98 can regulate IL1B, TNFA, MMP13 and COL2A1 genes expression indicating their important roles in the development of chondrocytes and osteocytes (73,74). ...
Background:
The details of molecular mechanisms underlying the differentiation of Mesenchymal Stem Cells (MSCs) into specific lineages are not well understood.
Objectives:
We aimed to construct the interactome network and topology analysis of bone marrow mesenchymal stem cell of CAGE data. Applying the enrichment results, we wanted to introduce the common genes and hub-microRNA and hub-genes of these giant network.
Materials and methods:
In this study, we constructed gene regulatory networks for each non-mesenchymal cell lineage according to their gene expression profiles obtained from FANTOM5 database. The putative interactions of TF-gene and protein-protein were determined using TRED, STRING, HPRD and GeneMANIA servers. In parallel, a regulatory network including corresponding miRNAs and total differentially expressed genes (DEGs) was constructed for each cell lineage.
Results:
The results indicated that analysis of networks' topology can significantly distinguish the hub regulatory genes and miRNAs involved in the differentiation of MSCs. The functional annotation of identified hub genes and miRNAs revealed that several signal transduction pathways i.e. AKT, WNT and TGFβ and cell proliferation related pathways play a pivotal role in the regulation of MSCs differentiation. We also classified cell lineages into two groups based on their predicted miRNA profiles.
Conclusions:
In conclusion, we found a number of hub genes and miRNAs which seem to have key regulatory functions during differentiation of MSCs. Our results also introduce a number of new regulatory genes and miRNAs which can be considered as the new candidates for genetic manipulation of MSCs in vitro.
... Convincing evidence indicates that BMSCs may be critically involved in bone differentiation and tissue regeneration [20,21]; hence understanding the osteogenic differentiation mechanism of BMSCs is important to provide new insight for increasing treatment options. Additional studies have suggested that osteogenic differentiation and bone remodeling processes are regulated by miRNAs [22][23][24]. ...
Trauma‐induced osteonecrosis of the femoral head (TIONFH) is characterized by femoral head collapse accompanied by degenerative changes of the hip. We previously reported that miR‐93‐5p expression is abnormally high in TIONFH patients, but the role of miR‐93‐5p in the TIONFH process remains unclear. Herein, we investigated the role of miR‐93‐5p in TIONFH in a rabbit model. Bone marrow mesenchymal stem cells (BMSCs) were used for both in vivo and in vitro experiments. A rabbit model of TIONFH was injected with BMSCs transfected with miR‐93‐5p inhibitor. In addition, both an miR‐93‐5p mimic and negative control were transfected into BMSCs. Expression of miR‐93‐5p was significantly increased in the model group compared with control samples. An miR‐93‐5p inhibitor induced the expression of BMP‐2 and ALP. Furthermore, expression of osteogenesis‐related markers (BMP‐2, OPN, RUNX‐2, and Osterix) was higher in the miR‐93‐5p inhibitor group, as revealed by qPCR and western blotting. Additionally, in vitro experimentation revealed that an miR‐93‐5p mimic decreased BMP‐2 and OPG expression, but increased RANKL expression. In summary, the miR‐93‐5p inhibitor could promote osteogenic differentiation by increasing BMP‐2 expression during the development of TIONFH. Thus, miR‐93‐5p may have potential as a therapeutic target for TIONF treatment.
... MicroRNAs (miRNAs) are a small class of noncoding single-stranded RNAs that are regulated at the posttranscriptional level by inhibiting mRNA translation or promoting mRNA degradation [8]. Many experimental data have indicated that miRNAs play a crucial regulatory role in the chondrogenic differentiation of MSCs [9][10][11]. By the regulation of specific genes, several miRNAs (miR-145 [12], miR-365 [13], and miR-218 [14]) have been shown to modulate chondrogenic differentiation of MSCs. ...
Cartilage injury of the knee joint is very common. Due to the limited self-healing ability of articular cartilage, osteoarthritis is very likely to occur if left untreated. Bone marrow mesenchymal stem cells (BMMSCs) are widely used in the study of cartilage injury due to their low immunity and good amplification ability, but they still have disadvantages, such as heterogeneous undifferentiated cells. MicroRNAs can regulate the chondrogenic differentiation ability of MSCs by inhibiting or promoting mRNA translation and degradation. In this research, we primarily investigated the effect of microRNA-210-3p (miR-210-3p) on chondrogenic and adipogenic differentiation of BMMSCs in vitro. Our results demonstrate that miR-210-3p promoted chondrogenic differentiation and inhibited adipogenic differentiation of rat BMMSCs, which was related to the HIF-3α signalling pathway. Additionally, miR-210-3p promotes mRNA and protein levels of the chondrogenic expression genes COLII and SOX9 and inhibits mRNA and protein levels of the adipogenic expression genes PPARγ and LPL. Thus, miR-210-3p combined with BMMSCs is a candidate for future clinical applications in cartilage regeneration and could represent a promising new therapeutic target for OA.
... Но, если Sox9 связан с хондрогенной дифференцировкой и усиливает активность экспрессии гена коллагена II типа (Coll2) и других маркеров начала дифференцировки, то Runx2 вовлечен в гипертрофическое созревание клеток. Кроме того, Sox9 ингибирует экспрессию Runx2 [1,[4][5][6]. ...
The authors studied the influence of the combination of TGF? and IGF growth factors, as well as the differentiation time, on the induction of MSC chondrogenesis in vitro. It is proved that MSCs located in 2D and 3D systems, when exposed to TGF?/ IGF, showed the signs of early chondroblast-like cells in 7 days. The TGF?/ IGF used for the induction of MSCs is more preferred, because it results in a more pronounced hypertrophic-suppression effect. The absence of significant differences in gene expression (excepting Sox9) on the 7th and 21st days of chondrogenic differentiation allows the process to be reduced in vitro to 7 days.
... containing a particular set of protein families from intracellular compartments (27,28). MSc-derived exosomes have been investigated in different disease models and have exhibited therapeutic potential in managing stroke, Parkinson's disease and OA (29)(30)(31)(32)(33). MicroRNAs (miRNAs or miRs), including miR-222, miR-140 and miR-381, regulate chondrogenesis and cartilage degeneration (34)(35)(36). Previous studies have demonstrated that miR-140 plays essential roles in regulating cartilage homeostasis and development by enhancing the expression of Sox9 and aggrecan (AcAN) (37)(38)(39). ...
Osteoarthritis (OA) is a common joint disorder, and the restoration of the impaired cartilage remains a main concern for researchers and clinicians. MicroRNAs (miRNAs or miRs) play crucial roles in the pathogenesis of OA. The present study examined the therapeutic efficacy of exosomal‑miR‑140‑5p for the treatment of OA using dental pulp stem cells (DPSCs). The findings indicated that the exosomal burden of miR‑140‑5p was substantially increased following the transfection of DPSCs with miR‑140‑5p mimic. The administration of DPSC‑derived exosomes promoted chondrocyte‑related mRNA expression, including aggrecan, Col2α1 and Sox9, in interleukin (IL)‑1β‑treated human chondrocytes. This effect was substantially enhanced by miR‑140‑5p‑enriched exosomes. The results further revealed that miR‑140‑5p‑enriched exosomes induced a more significant reduction in IL‑1β‑induced chondrocyte apoptosis than the DPSC‑derived exosomes. Mechanistically, it was found that miR‑140‑enriched DPSC‑derived exosomes exerted anti‑apoptotic effects, probably by regulating the expression levels of apoptosis‑related proteins. Furthermore, multiple administrations of miR‑140‑5p‑enriched exosomes substantially improved knee joint conditions in a rat model of OA. Collectively, the data of the present study suggest that exosomes derived from genetically modified DPSCs may prove to be a potential strategy for the treatment of OA.
... With nanoporosity, these pathways were not differentially solicited (Table S8), a finding that is consistent with the lesser inflammatory propensity of the nanoporous surface [22] and that may in part contribute to the overall improvement in bone healing under all loading conditions tested. Finally, nanotopography modulates the expression of some miRNAs during the bone healing [40], such as miR1224, which is implicated in osteolytic bone metastasis [41], and miR140, a regulator of osteogenesis and chondrogenesis [42,43] (Table S9). ...
Nanoscale surface modifications influence peri-implant cell fate decisions and implant loading generates local tissue deformation, both of which will invariably impact bone healing. The objective of this study is to determine how loading affects healing around implants with nanotopography. Implants with a nanoporous surface were placed in over-sized osteotomies in rat tibiae and held stable by a system that permits controlled loading. Three regimens were applied: (a) no loading, (b) one daily loading session with a force of 1.5N, and (c) two such daily sessions. At 7 days post implantation, animals were sacrificed for histomorphometric and DNA microarray analyses. Implants subjected to no loading or only one daily loading session achieved high bone implant contact (BIC), bone implant distance (BID) and bone formation area near the implant (BFAt) values, while those subjected to two daily loading sessions showed less BFAt and BIC and more BID. Gene expression profiles differed between all groups mainly in unidentified genes, and no modulation of genes associated with inflammatory pathways was detected. These results indicate that implants with nanotopography can achieve a high level of bone formation even under micromotion and limit the inflammatory response to the implant surface.
... MicroRNAs (miRNAs) are a group of small, noncoding single-stranded RNA with a tissue or developmental stage specificity, which plays a regulatory role through posttranscriptional binding to specific sequences of target genes. Several miRNAs were verified to take part in the processes of cartilage differentiation and degradation (Dong et al. 2012;Kobayashi et al. 2008). Our previous study revealed that miR-23a-3p was downregulated during chondrogenic differentiation. ...
Cartilage generation and degradation are controlled by miRNAs. Our previous study showed miR-23a-3p was downregulated during chondrogenic differentiation in chondrogenic human adipose-derived mesenchymal stem cells (hADSCs). In the present study, we explored the function of miR-23a-3p in chondrogenesis differentiation. The role of miR-23a-3p in chondrogenic differentiation potential of hADSCs was assessed by Alcian blue staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. We show that miR-23a-3p suppressed the chondrogenic differentiation of hADSCs. LncRNA SNHG5 interacted with miR-23a-3p, and suppression or overexpression of SNHG5 correlates with inhibition and promotion of hADSC chondrogenic differentiation, respectively. We have determined that SNHG5 can sponge miR-23a-3p to regulate the expression of SOX6/SOX5, transcription factors that play essential roles in chondrocyte differentiation. Furthermore, the overexpression of SNHG5 activates the JNK/MAPK/ERK pathway. In conclusion, miR-23a-3p regulated by lncRNA SNHG5 suppresses the chondrogenic differentiation of human adipose-derived stem cells via targeting SOX6/SOX5.
... Osteoporosis and osteoarthritis are orthopedic disorders that affect millions of elderly people worldwide and are characterized by bone nonunion, loss, and defects that lead to impaired bone formation and bone deterioration [1]. Stimulating bone formation is a potential therapeutic strategy for the treatment of these conditions. ...
Osteoporosis and osteoarthritis are orthopedic disorders that affect millions of elderly people worldwide; stimulation of bone formation is a potential therapeutic strategy for the treatment of these conditions. As the only bone-forming cells, osteoblasts play a key role in bone reconstruction. The microRNA miR-17-3p is downregulated during osteogenic differentiation of human bone marrow mesenchymal stem cells, but its precise role in this process is unknown. Here, we investigated the role of miR-17-3p in osteoblast differentiation. An in vitro model of osteogenesis was established by treating MC3T3-E1 murine pre-osteoblast cells with bone morphogenetic protein 2 (BMP2). The expression of miR-17-3p in BMP2-induced MC3T3-E1 cells was detected by reverse transcription-quantitative PCR, and its effects on cells transfected with miR-17-3p mimic or inhibitor were evaluated by Alizarin Red staining, alkaline phosphatase (ALP) activity assay, and by detection of osteoblast markers including the ALP, collagen type I α1 chain, and osteopontin genes. Bioinformatics analysis was carried out to identify putative target genes of miR-17-3p, and the luciferase reporter assay was used for functional validation. Rescue experiments were performed to determine whether SRY-box transcription factor 6 (Sox6) plays a role in the regulation of osteoblast differentiation by miR-17-3p. We report that miR-17-3p was downregulated upon BMP2-induced osteoblast differentiation in MC3T3-E1 cells, and this was accompanied by decreased differentiation and mineralization, ALP activity, and expression of osteogenesis-related genes. Sox6 was confirmed to be a target gene of miR-17-3p in osteoblasts, and the inhibitory effect of miR-17-3p on osteoblast differentiation was observed to occur via Sox6. These results suggest the existence of a novel mechanism underlying miRNA-mediated regulation of osteogenesis, which has potential implications for the treatment of orthopedic disorders.
... [18][19][20] According to these studies, it has been reported that during the osteogenic differentiation of some stem cells, the expression level of miR-141, -200a, -196a, -29b, -2861, -3960, -21, -335-5p, and -2861 will be increased. 21,22 Also, the expression level of miR-10a, -10b, -19b, -9-3p, -124a, -181a, -146a, -26a, -133, -135, -141, and -200a will also be decreased. 23,24 Depending on their target proteins, microRNAs can be effective in various signal pathways, including osteogenesis signaling. ...
Tissue engineering using new strategies has become a growing and promising method for treating large tissue lesions in the body. On the other hand, microRNAs (miRNAs), which are small non‐coding regulatory RNAs, are a new class of genetic materials that can have effective pharmacological roles. The combination of these two themes has created promising prospects for the treatment of diseases. Herein, human induced pluripotent stem cells (iPSCs) were transduced with miRNA‐2861 and then the osteogenic differentiation potential of transduced iPSCs and non‐transduced iPSCs was investigated while cultured on the electrospun poly lactic‐co‐glycolic acid (PLGA) nanofibrous scaffold and culture plate. MiR‐2861‐transduced iPSCs showed a significantly higher viability, mineralization, alkaline phosphatase (ALP) activity, calcium content, and bone‐related gene expression in comparison with those iPSCs that non‐transduced. The results also indicated that this increase is improved when miR‐2861 transduced iPSCs are cultured on the PLGA nanofibrous scaffold synergistically. This synergy was also confirmed by the results obtained from of Western blot analysis. It can be concluded that, miR‐2861, by negative regulation of those proteins that decrease/inhibit osteogenic differentiation and PLGA nanofibrous scaffold by preparation of a suitable artificial extracellular matrix, have a great positive impact in improving iPSCs osteogenic differentiation potential and this blend can be proposed to use in bone tissue engineering application.
... The mechanisms by which the transcription or regulatory factors are involved in MSC differentiation towards the osteogenic lineage have been proposed in many studies [17]. Among those, miRNAs that remain well conserved in different species have recently been revealed as important regulators in the lineage commitment of MSC, osteogenic differentiation, and bone formation [18][19][20]. Furthermore, the involvement of miRNAs in the mechanotransduction pathways of osteogenic differentiation has also been studied [21][22][23]. Our previous studies showed that miRNA-132-3p was obviously increased in both bone tissue and osteoblast cells in gravitational mechanical unloading [24]. ...
Background:
Skeletal unloading can induce severe disuse osteopenia that often occurs in spaceflight astronauts or in patients subjected to prolonged bed-rest or immobility. Previously, we revealed a mechano-sensitive factor, miRNA-132-3p, that is closely related to the osteoblast function. The aim of this study was to investigate whether miRNA-132-3p could be an effective target for treating disuse osteopenia.
Methods:
The 2D-clinostat device and the hindlimb-unloaded (HU) model were used to copy the mechanical unloading condition at the cellular and animal levels, respectively. Mimics or inhibitors of miRNA-132-3p were used to interfere with the expression of miRNA-132-3p in bone marrow-derived mesenchymal stem cells (BMSCs) in vitro for analyzing the effects on osteogenic differentiation. The special in vivo antagonists of miRNA-132-3p was delivered to the bone formation regions of HU mice for treating disuse osteopenia by a bone-targeted (AspSerSer)6-cationic liposome system. The bone mass, microstructure, and strength of the hindlimb bone tissue were analyzed for evaluating the therapeutic effect in vivo.
Results:
miRNA-132-3p expression was declined under normal conditions and increased under gravitational mechanical unloading conditions during osteogenic differentiation of BMSCs in vitro. The upregulation of miRNA-132-3p expression resulted in the inhibition of osteogenic differentiation, whereas the downregulation of miRNA-132-3p expression enhanced osteogenic differentiation. The inhibition of miRNA-132-3p expression was able to attenuate the negative effects of mechanical unloading on BMSC osteogenic differentiation. Most importantly, the targeted silencing of miRNA-132-3p expression in the bone tissues could effectively preserve bone mass, microstructure, and strength by promoting osteogenic differentiation and osteogenesis in HU mice.
Conclusion:
The overexpression of miRNA-132-3p induced by mechanical unloading is disadvantageous for BMSC osteogenic differentiation and osteogenesis. Targeted silencing of miRNA-132-3p expression presents a potential therapeutic target for the prevention and treatment of disuse osteoporosis.
... Genetic variants in miRNAs gene promoters or genes have been associated with susceptibility to different diseases, such as cancer, cardiovascular disease and autoimmune diseases, through changing mature miRNAs production and subsequent function [14][15][16]. expression contributes to pathology of cartilage diseases, including OA [17,18]. It has been shown that many miRNAs with aberrant expression levels in OA tissues, regulate inflammatory and pain pathways, as well as pathways involved in cartilage degradation, whereas specific circulating miRNAs in serum or synovial fluid of OA patients have been suggested as potential OA biomarkers [19][20][21][22]. ...
Purpose:
MiR-146a acts as a negative inflammatory mediator in different diseases and has been implicated in osteoarthritis (OA) pathogenesis. In our study, we investigated the association between miR-SNP rs2910164 and OA susceptibility and its role on the expression of miR-146a, inflammatory and catabolic mediators in osteoarthritic chondrocytes.
Materials and methods:
Genetic association analysis was performed in 1688 knee OA patients and healthy individuals of Greek origin. Genomic DNA was extracted from blood and genotyped for rs2910164 (G > C) using Restriction-Fragment Length Polymorphism (RFLP). Total RNA was extracted from chondrocytes of 18 OA patients and miR-146a, IL-1 Receptor-Associated Kinase 1 (IRAK-1), TNF Receptor-Associated Factor 6 (TRAF-6), A Disintegrin and Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS-5), Matrix Metalloproteinase-13 (MMP-13), Interleukin-6 (IL-6), Interleukin-1 Beta (IL-1β) and Tumor Necrosis Factor-Alpha (TNF-α) expression was evaluated using quantitative Real-Time PCR (qRT-PCR).
Results:
OA patients carrying rs2910164-GC and CC genotypes did not have an increased risk for OA development compared to GG genotype carriers. MiR-146a expression in OA chondrocytes was significantly lower in patients with rs2910164-GC genotype than in the GG carriers. OA patients carrying the rs2910164-GC genotype in their chondrocytes exhibited increased IRAK-1, TRAF-6, MMP-13, IL-1β and IL-6 expression levels compared with rs2910164-GG carriers.
Conclusion:
We demonstrate, for the first time, that miR-SNP rs2910164 in miR-146a gene is associated with reduced miR-146a and increased inflammatory and catabolic mediators' expression in OA chondrocytes. Our data imply that genetic variations in miRNAs linked to OA pathogenesis may regulate their expression levels, suggesting new therapeutic strategies for patients with cartilage diseases.
... With respect to the joint, the biogenesis and activity of miRNAs respond to mechanical load (Guo et al., 2015;Le, Swinger, & Clark, 2013), aging of mesenchymal stem cells (He, Eberhart, & Postlethwait, 2009), and hypoxia (Nallamshetty, Chan, & Loscalzo, 2013). These properties appear to facilitate the "micromanagement" of the vertebrate skeleton in all species from fish to mammals (Dong, Yang, Guo, & Kang, 2012;He et al., 2009). Periods of rapid evolutionary change, such as at the origins of vertebrates and placental mammals, are believed to include significant proliferation and activity of miRNA (Hertel et al., 2006). ...
Developmental osteogenesis and the pathologies associated with tissues that normally are mineralized are active areas of research. All of the basic cell types of skeletal tissue evolved in early aquatic vertebrates. Their characteristics, transcription factors, and signaling pathways have been conserved, even as they adapted to the challenge imposed by gravity in the transition to terrestrial existence. The response to excess mechanical stress (among other factors) can be expressed in the pathologic phenotype described as osteoarthritis (OA). Osteoarthritis is mediated by epigenetic modification of the same conserved developmental gene networks, rather than by gene mutations or new chemical signaling pathways. Thus, these responses have their evolutionary roots in morphogenesis. Epigenetic channeling and heterochrony, orchestrated primarily by microRNAs, maintain the sequence of these responses, while allowing variation in their timing that depends at least partly on the life history of the individual. This article is protected by copyright. All rights reserved.
... For example, it has been demonstrated that overexpression of miR-206, miR-133, miR-135 and miR125b inhibit osteogenic differentiation. On the other hand, the expression of cluster miR-17-92, miR-196a and miR-2861 has the opposite effect, enhancing osteogenic differentiation [100]. ...
Bone injuries and diseases constitute a burden both socially and economically, as the consequences of a lack of effective treatments affect both the patients’ quality of life and the costs on the health systems. This impended need has led the research community’s efforts to establish efficacious bone tissue engineering solutions. There has been a recent focus on the use of biomaterial-based nanoparticles for the delivery of therapeutic factors. Among the biomaterials being considered to date, calcium phosphates have emerged as one of the most promising materials for bone repair applications due to their osteoconductivity, osteoinductivity and their ability to be resorbed in the body. Calcium phosphate nanoparticles have received particular attention as non-viral vectors for gene therapy, as factors such as plasmid DNAs, microRNAs (miRNA) and silencing RNA (siRNAs) can be easily incorporated on their surface. Calcium phosphate nanoparticles loaded with therapeutic factors have also been delivered to the site of bone injury using scaffolds and hydrogels. This review provides an extensive overview of the current state-of-the-art relating to the design and synthesis of calcium phosphate nanoparticles as carriers for therapeutic factors, the mechanisms of therapeutic factors’ loading and release, and their application in bone tissue engineering.
... MicroRNAs (miRNAs) are non-coding endogenous RNAs composed of short nucleotide sequences, 20-24 nucleotides, that act in the post-transcriptional regulation of gene expression [1]. As soon as miRNA activity was confirmed in humans [2,3], significant attention was generated on their clinical application, because of their impact on critical cellular activities, such as metabolism [4], differentiation/ proliferation [5,6] and programmed cell death [6]. Several studies have described the influence of miRNAs on the onset and progression of diseases, including cancer [7][8][9], neurodegenerative disorders [10,11], cardiovascular diseases [12][13][14], and other pathologies [15][16][17]. ...
MicroRNAs (miRNAs) are attracting a growing interest in the scientific community due to their central role in the etiology of major diseases. On the other hand, nanoparticle carriers offer unprecedented opportunities for cell specific controlled delivery of miRNAs for therapeutic purposes. This review critically discusses the use of nanoparticles for the delivery of miRNA-based therapeutics in the treatment of cancer and neurodegenerative disorders and for tissue regeneration. A fresh perspective is presented on the design and characterization of nanocarriers to accelerate translation from basic research to clinical application of miRNA-nanoparticles. Main challenges in the engineering of miRNA-loaded nanoparticles are discussed, and key application examples are highlighted to underline their therapeutic potential for effective and personalized medicine.
... The present study is based on the hypothesis that both the stromal origin cells of C2C12 and hMSc exhibit some degree of plasticity to support the genetic reprogramming [17,18]. The hypothetical pathway, leading to neural-like cells from the committed adult cells (C2C12), can be considered as dedifferentiation to achieve stemness and transdifferentiation to generate non-muscular cell type. ...
A number of bioengineering strategies, using biophysical stimulation, are being explored to guide the human mesenchymal stem cells (hMScs) into different lineages. In this context, we have limited understanding on the transdifferentiation of matured cells to another functional-cell type, when grown with stem cells, in a constrained cellular microenvironment under biophysical stimulation. While addressing such aspects, the present work reports the influence of the electric field (EF) stimulation on the phenotypic and functionality modulation of the coculture of murine myoblasts (C2C12) with hMScs [hMSc:C2C12=1:10] in a custom designed polymethylmethacrylate (PMMA) based microfluidic device with in-built metal electrodes. The quantitative and qualitative analysis of the immunofluorescence study confirms that the cocultured cells in the conditioned medium with astrocytic feed, exhibit differentiation towards neural-committed cells under biophysical stimulation in the range of the endogenous physiological electric field strength (8 ± 0.06 mV/mm). The control experiments using similar culture protocols revealed that while C2C12 monoculture exhibited myotube-like fused structures, the hMScs exhibited the neurosphere-like clusters with SOX2, nestin, βIII-tubulin expression. The electrophysiological study indicates the significant role of intercellular calcium signalling among the differentiated cells towards transdifferentiation. Furthermore, the depolarization induced calcium influx strongly supports neural-like behaviour for the electric field stimulated cells in coculture. The intriguing results are explained in terms of the paracrine signalling among the transdifferentiated cells in the electric field stimulated cellular microenvironment. In summary, the present study establishes the potential for neurogenesis on-chip for the coculture of hMSc and C2C12 cells under tailored electric field stimulation, in vitro.
... MiRNAs play an important role in cartilage regenerative medicine [31,32]. It has been reported that MiR-193b-3p promotes hMSC chondrogenesis and metabolism in chondrocytes [15]. ...
Stable cartilage regeneration has always been a challenge in both tissue engineering research and clinical practice. This study explored the feasibility of using a chondrocyte sheet technique stimulated by microRNAs to regenerate cartilage. We tested the involvement of hsa-miR-193b-3p in the microtia patient remnant auricular chondrocyte extracellular matrix (ECM). We observed in vitro chondrocyte proliferation, ECM synthesis, as well as the increase in the expression of type II collagen (COL2A1) and decrease in the expression of matrix metalloproteinase 16 (MMP16) of the chondrocyte sheets. COL2A1 deposition and MMP16 degradation of regenerative cartilage tissue were examined in vivo. A dual-luciferase reporter showed that the MMP16 gene was the direct target of miR-193b-3p. These results suggested that miR-193b-3p promotes chondrocyte sheet ECM synthesis by inhibiting MMP16. Since the evidence suggests that MMP16 is a critical regulator of chondrocyte ECM, this finding points the way towards a method that both strengthens the ECM and inhibits MMPs.
... miR-146a/b is found to be a novel molecule for reversing excessive differentiation of osteoclasts by inactivating the NF-κB molecule, an activator of osteoclast precursor cells, which eventually led to reduced joint destruction in arthritic mice [151]. Overexpression or synthetic transfer of miR-145 in chondrocytes can potentially be used for treating cartilage degradation [152]. Many miRNAs that target Runx2 repressors are potential therapeutic candidates: miR-2861 targets HDAC5; miR-29b targets HDAC4 and miR-15b targets E3 ubiquitin ligase; and others [153]. ...
... This is while during negative osteogenesis, Runx2 or its co-activators can be targeted by miRs. 32 For example, miR-29 is one type of miRs which its role in bone formation has been determined. Actually, miR-29 can stimulate osteogenic differentiation by targeting negative regulators on the related signaling pathway. ...
... Osteogenesis in bone regeneration and remodeling is coupled with angiogenesis by osteo-angiogenic factors which is released by pre-osteoblast/osteoclast and osteoblast/clast cells [1]. It has been shown that miRNAs are involved in physiologic bone and vessel formation by targeting various transcription factors in stem cells, osteoblasts, osteoclasts, and chondrocytes [45,46]. In addition, increasing evidence demonstrates miRNAs role in cardiovascular development and angiogenesis in tumor [47]. ...
MicroRNAs (miRNAs) as a newly founded and thriving non-coding endogenous class of molecules which regulate many cellular pathways after transcription have been extensively investigated in regenerative medicine. In this systematic review, we sought to analyze miRNAs-mediated therapeutic approaches for influencing angiogenesis in bone tissue/bone regeneration. An electronic search in MEDLINE, Scopus, EMBASE, Cochrane library, web of science, and google scholar with no time limit were done on English publications. All types of original articles which a miRNA for angiogenesis in bone regeneration were included in our review. In the process of reviewing, we used PRISMA guideline and, SYRCLE’s and science in risk assessment and policy tools for analyzing risk of bias. Among 751 initial retrieved records, 16 studies met the inclusion criteria and were fully assessed in this review. 275 miRNAs, one miRNA 195~497 cluster, and one Cysteine-rich 61 short hairpin RNA were differentially expressed during bone regeneration with 24 predicted targets reported in these studies. Among these miRNAs, miRNA-7b, -9, -21, -26a, -27a, -210, -378, -195~497 cluster, -378 and -675 positively promoted both angiogenesis and osteogenesis, whereas miRNA-10a, -222 and -494 inhibited both processes. The most common target was vasculoendothelial growth factor-signaling pathway. Recent evidence has demonstrated that miRNAs actively participated in angio-osteogenic coupling that can improve their therapeutic potentials for the treatment of bone-related diseases and bone regeneration. However, there is still need for further research to unravel the exact mechanisms.
... Numerous regulatory pathways and stimulus of specific factors are implicated in the progression of osteogenesis [5]. Accumulating evidence suggests that osteoblastic induction and differentiation are also regulated by post-transcriptional mechanisms, partly through temporarily-expressed microRNAs (miRNAs) [6]. miR-NAs have been shown to be involved in diverse biological processes including cellular differentiation [7], proliferation [8] and apoptosis [9]. ...
Osteogenesis is a complex process which relies on the coordination of signals and transcription factors. Recent evidence indicates that microRNAs (miRNAs) act as important post-transcriptional regulators in a large number of biological processes including osteoblast differentiation. In this study, we investigated the expression and biological effect of miR-125a-3p during osteogenic differentiation of human adipose derived mesenchymal stem cells (hADSCs). We observed an obvious decrease in miR-125a-3p level during osteogenic differentiation. By using gain- and loss-of function experiments, we noticed that miR-125a-3p could suppress the osteogenic differentiation of hADSCs. Moreover, miR-125a-3p over-expression in hADSCs by transfection with miR-125a-3p mimics significantly inhibited cell proliferation by MTT. Flow cytometry analysis further demonstrated that forced expression of miR-125a-3p induced cell cycle G1/S phase arrest and apoptosis. In addition, we performed bioinformatic analysis, luciferase reporter assay and western blot to confirm that miR-125a-3p could regulate Smad4 and Jak1 expression negatively. Meanwhile, Smad4 and Jak1 were up-regulated after osteogenic differentiation and the down-regulation of endogenous Smad4 and Jak1 suppressed the osteogenic differentiation of hADSCs. Taken together, these data indicated that miR-125a-3p is Smad4 and Jak1 regulator, and it has a crucially physiological function in osteogenic differentiation of hADSCs.
... It is now well established that miRNAs are physiologically relevant to all steps of bone as well as blood vessel formation during embryonic development and in maintenance during adulthood [63]. OsteomiRs have been identified to regulate chondrocyte, osteoblast, and osteoclast differentiation by positively targeting the principal osteogenic transcription factors and signaling molecules of osteogenesis [5,38,[64][65][66][67][68][69]. In addition to regulating MSC commitment-i.e., the differentiation of precursor cells into chondrocytic and osteoblastic lineages-several studies showed that miRNAs also contribute to the maturation and function of these cells, suggesting also important roles in bone regeneration. ...
Bone formation and regeneration is a multistep complex process crucially determined by the formation of blood vessels in the growth plate region. This is preceded by the expression of growth factors, notably the vascular endothelial growth factor (VEGF), secreted by osteogenic cells, as well as the corresponding response of endothelial cells, although the exact mechanisms remain to be clarified. Thereby, coordinated coupling between osteogenesis and angiogenesis is initiated and sustained. The precise interplay of these two fundamental processes is crucial during times of rapid bone growth or fracture repair in adults. Deviations in this balance might lead to pathologic conditions such as osteoarthritis and ectopic bone formation. Besides VEGF, the recently discovered important regulatory and modifying functions of microRNAs also support this key mechanism. These comprise two principal categories of microRNAs that were identified with specific functions in bone formation (osteomiRs) and/or angiogenesis (angiomiRs). However, as hypoxia is a major driving force behind bone angiogenesis, a third group involved in this process is represented by hypoxia-inducible microRNAs (hypoxamiRs). This review was focused on the identification of microRNAs that were found to have an active role in osteogenesis as well as angiogenesis to date that were termed “CouplingmiRs (CPLGmiRs)”. Outlined representatives therefore represent microRNAs that already have been associated with an active role in osteogenic-angiogenic coupling or are presumed to have its potential. Elucidation of the molecular mechanisms governing bone angiogenesis are of great relevance for improving therapeutic options in bone regeneration, tissue-engineering, and the treatment of bone-related diseases.
... A recent study demonstrates that miRNAs are involved in various cellular processes, such as cell proliferation, migration, differentiation, and apoptosis (10). Increasing evidence indicates that miRNAs regulate the differentiation and function of chondrocytes, osteoblasts, and osteoclasts (11). These findings suggest that miRNAs act as key mediators in the processes of bone formation, resorption, remodeling, and repair (12). ...
Bone fracture is a common medical condition, which may occur due to traumatic injury or disease-related conditions. Evidence suggests that microRNAs (miRNAs) can regulate osteoblast differentiation and function. In this study, we explored the effects and mechanism of miR-221 on the growth and migration of osteoblasts using MC3T3-E1 cells. The expression levels of miR-221 in the different groups were measured by qRT-PCR. Then, miR-221 mimic and inhibitor were transfected into MC3T3-E1 cells, and cell viability and migration were measured using the CCK-8 assay and the Transwell migration assay. Additionally, the expression levels of differentiation-related factors (Runx2 and Ocn) and ZFPM2 were measured by qRT-PCR. Western blot was used to measure the expression of cell cycle-related proteins, epithelial-mesenchymal transition (EMT)-related proteins, ZFPM2, and Wnt/Notch, and Smad signaling pathway proteins. miR-221 was significantly up-regulated in the patients with lumbar compression fracture (LCM) and trochanteric fracture (TF). miR-221 promoted ALP, Runx2, and OPN expressions in MC3T3-E1 cells. miR-221 overexpression significantly increased cell proliferation, migration, differentiation, and matrix mineralization, whereas suppression of miR-221 reversed these effects. Additionally, the results displayed that ZFPM2 was a direct target gene of miR-221, and overexpression of ZFPM2 reversed the promoting effects of miR-221 overexpression on osteoblasts. Mechanistic study revealed that overexpression of miR-221 inactivated the Wnt/Notch and Smad signaling pathways by regulating ZFPM2 expression. We drew the conclusions that miR-221 overexpression promoted osteoblast proliferation, migration, and differentiation by regulation of ZFPM2 expression and deactivating the Wnt/Notch and Smad signaling pathways.
... They are key regulators of numerous pathways and cellular processes. In bone, a number of miRNAs have been reported to be regulators of osteoblast differentiation [miR29a, 29b, 141, 200a, 210, 206, 133, 135, 125b, 26a, 2861, and 196a [93,94]] and chondrocyte differentiation [miR18a, 199a, 146a, 222, 140, and 27b [94]]. ...
Information about the molecular mechanisms leading to the activation of the osteoclast is relatively limited. While there is compelling evidence that the signaling mechanisms of Src and integrin β3 are essential for osteoclast activation, the regulation of these two signaling mechanisms is not fully understood. In this review, evidence supporting a novel regulatory axis of osteoclast activation that plays an upstream regulatory role in both the Src and integrin β3 signaling during osteoclast activation is discussed. This regulatory axis contains three unique components: a structurally unique transmembrane protein-tyrosine phosphatase, PTP-oc, EphA4, and miR17. In the first component, PTP-oc activates the Src signaling through dephosphorylation of the inhibitory tyr-527 of Src. This in turn activates the integrin β3 signaling, enhances the JNK2/NFκB signaling, promotes the ITAM/Syk signaling, and suppresses the ITIM/Shp1 signaling; the consequence of which is activation of the osteoclast. In the second component, EphA4 inhibits osteoclast activity by suppressing the integrin β3 signaling. PTP-oc relieves the suppressive actions of EphA4 by directly dephosphorylating EphA4. In the third component, PTP-oc expression is negatively regulated by miR17. Accordingly, suppression of miR17 during osteoclast activation upregulates the PTP-oc signaling and suppresses the EphA4 signaling, resulting in the activation of the osteoclast. This regulatory axis is unique, in that each of the three components acts to exert suppressive action on their respective immediate downstream inhibitory step. Because the final downstream event is the EphA4-mediated inhibition of osteoclast activation, the overall effect of this mechanism is the stimulation of osteoclast activity.
... by targeting Runt-related transcription factor 2 differentiation and bone formation by targeting negative regulators of osteogenesis, or negatively regulate it by targeting important osteogenic factors (19)(20)(21)(22). In particular, miR-153 has been proven to regulate cell proliferation and differentiation in osteosarcoma, prostate cancer, gastric cancer and venous smooth muscle cell lines (23)(24)(25)(26). ...
Osteoporosis has become one of the most serious public health problems. Icariin, miR-153 and Runt-related transcription factor 2 (Runx2) have been demonstrated to regulate cell proliferation and differentiation in multiple cells. The aim of the present experiments was to investigate the potential mechanism underlying osteoblast differentiation and cell proliferation of MC3T3-E1 cells treated with icariin. Cell Counting kit-8, alkaline phosphatase (ALP) activity and aliz-arin red S assays, as well as reverse transcription-quantitative polymerase chain reaction and western blot analysis, were performed to examine whether icariin promoted osteoblast differentiation and cell proliferation in MC3T3-E1 cells. Subsequently, miR-153 target and pathway prediction, and functional analysis were assessed. The results demonstrated that icariin promoted proliferation, mineral content and ALP activity in MC3T3-E1 cells. In addition, miR-153 and Runx2 expression levels were increased following treatment with icariin. Luciferase assay revealed that miR-153 significantly upregulate the luciferase activity of wild-type (Wt) Runx2 3'-untranslated region. Furthermore, the group treated with a combination of miR-153 mimics and icariin exhibited a significantly higher expression of Runx2 in comparison with the miR-153 mimic-treated alone group. Finally, icariin reversed the potential effect of miR-153 inhibitor in MC3T3-E1 cells. In conclusion, icariin exerted a strong osteoblast differentiation effect in MC3T3-E1 cells through the miR-153/Runx2 pathway. The current study provided evidence suggesting that icariin should be considered as an effective candidate for the management of osteoporosis.
... A growing number of evidence indicated that miRNAs are involved in the control of various basic cell functions [8]. Studied have indicated that miRNAs have an important role in the process of chondrogenesis and cartilage remodeling [9]. The aberrant miRNA expression profiles have been demonstrated to be associated with OA [10,11], and miRNAs acted as important regulators in OA development, as well as participated in inflammatory response [12,13]. ...
Background/aims:
Osteoarthritis (OA) as a degenerative disease is a major problem in ageing populations. To better understand the molecular mechanisms in the pathogenesis of OA, this study explored the role of microRNA (miR)-146a in the articular chondrocytes.
Methods:
The articular chondrocyte line ATDC5 was used to simulate inflammatory injury by LPS administration in vitro. Cell viability, apoptosis, mRNA expressions and productions of inflammatory factors were assessed, respectively. Mir-146a and Cxcr4 mRNA expressions were measured by qRT-PCR. Targeting effect of miR-146a on Cxcr4 3'UTR was assessed by luciferase activity analysis. Protein expression levels of CXCR4 and main factors in PI3K/AKT, Wnt/β-catenin signal pathways were measured by western blotting.
Results:
LPS exposure suppressed cell viability, prompted apoptosis of ATDC5 cells, and stimulated expression and release of inflammatory factors. MiR-146a was upregulated in LPS-induced cells. Overexpression of miR-146a further aggravated LPS-induced inflammatory injury, while it was reduced after miR-146a was knocked down. CXCR4 expression was negatively regulated by miR-146a. CXCR4 was a direct target of miR-146a and thus involved in regulatory effect of miR-146a on the injured chondrocytes, which was also related with phosphorylation levels of PI3K/AKT and expressions of Wnt/β-catenin signal factors.
Conclusion:
miR-146a promoted inflammatory response of articular chondrocytes via targeting CXCR4 and suppressing CXCR4 expression. Overexpression of CXCR4 could attenuate the inflammatory injury. Our findings provided novel evidence which might be useful for further studies exploring therapeutic approaches for OA via targeting miR-146a.
miRNAs represent appropriate candidates for treatment of several disorders. However, safe and efficient delivery of these small-sized transcripts has been challenging. Nanoparticle-based delivery of miRNAs has been used for treatment of a variety of disorders, particularly cancers as well as ischemic stroke and pulmonary fibrosis. The wide range application of this type of therapy is based on the important roles of miRNAs in the regulation of cell behavior in physiological and pathological conditions. Besides, the ability of miRNAs to inhibit or increase expression of several genes gives them the superiority over mRNA or siRNA-based therapies. Preparation of nanoparticles for miRNA delivery is mainly achieved through using protocols originally developed for drugs or other types of biomolecules. In brief, nanoparticle-based delivery of miRNAs is regarded as a solution for overcoming all challenges in the therapeutic application of miRNAs. Herein, we provide an overview of studies which used nanoparticles as delivery systems for facilitation of miRNAs entry into target cells for the therapeutic purposes. However, our knowledge about miRNA-loaded nanoparticles is limited, and it is expected that numerous therapeutic possibilities will be revealed for miRNA-loaded nanoparticles in future.
Slipped capital femoral epiphysis (SCFE) is a multifactorial disease caused by a number of factors, and each of them can be triggered in its occurrence. Modern research is increasingly pointing to the leading role of epigenetic structures in the origin and course of this group of diseases. In recent years, the most promising area is the study of the role of microRNAs as an epigenetic factor that plays a leading role in the pathogenesis of multifactorial diseases. Objective: to develop a methodology for genetic genealogical examination of patients with juvenile epiphysiolysis of the femoral head. Materials and Methods. The study material was 26 patients with SCFE (15 girls and 11 boys) in different periods of sexual development: prepuberty – 7 patients, induction of puberty – 12 patients, and puberty and postpuberty – 7 patients. A questionnaire developed by us was used for clinical and genealogical research of the patients. The material for the epigenetic study, namely the study of microRNA-21 expression in patients with SCFE, was venous blood. Results. The expression of microRNA-21 in the blood of patients with SCFE is different (average expression is 0.86 conventional units) from those of healthy donors (average 0.393 conventional units) of the same age (p<0.05). Analysis of dependence on the period of sexual development revealed statistical differences in indicators depending on the group (p<0.05). No dependence of the level of microRNA-21 expression in patients with SCFE on sex and the period before or after surgery (p>0.05) was revealed. The obtained data on the probability of the influence of hereditary factors on the occurrence of SCFE are statistically significant (p<0.05). Conclusions. Our results of epigenetic and clinical genealogical research of patients with SCFE showed a high probability of influence of hereditary and epigenetic factors on the occurrence and course of this disease.
Noncoding RNAs (ncRNAs) play an essential role in the control of target gene expression underlying various processes of bone cells, thus dysregulation of ncRNAs is involved in the pathogenesis and progression of bone disease. Development of novel RNA-based therapeutics for bone disorders holds the promise to improve the healing of bone. This chapter summarized the technologies used for ncRNA synthesis which were employed in the therapeutic studies of bone-related diseases in the recent 5 years. Viral vector and chemical synthesis approaches were briefly introduced, and the novel biotechnology that was manufacturing recombinant ncRNA agents through microbial fermentation was highlighted. The in vivo fermentation method could achieve an efficient, large-scale, and cost-effective production of recombinant or bioengineered ncRNAs (as much as tens of milligrams from 1 L of bacterial culture), and this approach may shift the paradigm to apply the natural, endogenous-similar biological ncRNA molecules for bone disease research and therapy.
Stem cell-derived extracellular vesicles (EVs) have shown great promise in the field of regenerative medicine and tissue engineering. Recently, human bone marrow-derived mesenchymal stem cell (BMSC)-derived EVs have been considered for bone tissue engineering applications. In this study, we evaluated the osteogenic capability of placental stem cell (PSC)-derived EVs and compared them to the well-characterized BMSC-derived EVs. EVs were extracted from three designated time points (0, 7, and 21 days) after osteogenic differentiation. The results showed that the PSC-derived EVs had much higher protein and lipid concentrations than EVs derived from BMSCs. The extracted EVs were characterized by observing their morphology and size distribution before utilizing next-generation sequencing to determine their microRNA (miRNA) profiles. A total of 306 miRNAs within the EVs were identified, of which 64 were significantly expressed in PSC-derived EVs that related to osteogenic differentiation. In vitro osteogenic differentiation study indicated the late-stage (21-day extracted)-derived EVs higher osteogenic enhancing capability when compared with the early stage-derived EVs. We demonstrated that EVs derived from PSCs could be a new source of EVs for bone tissue engineering applications.
Background: Bone fracture is a common medical condition. Evidence suggested that long noncoding RNAs (lncRNAs) could regulate the bio-function in osteoblast. In this study, we explored the role and mechanism of lncRNA X-inactive specific transcript (XIST) on the proliferation, apoptosis, and differentiation of osteoblasts using MC3T3-E1 cells. Methods: Expression of XIST, microRNA-203-3p (miR-203-3p), and zinc finger protein multitype 2 (ZFPM2) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis of MC3T3-E1 cells were measured using the Cell Counting Kit-8 (CCK-8) and the flow cytometry. Western blot was used to measure the expression of cell cycle-related proteins, apoptosis-related proteins, and ZFPM2. Levels of differentiation-related factors were measured by qRT-PCR, western blot, and alkaline phosphatase (ALP) kit. Target interaction between miR-203-3p and XIST or ZFPM2 was predicted through bioinformatics analysis and verified by dual-luciferase reporter, RNA immunoprecipitation (RIP) assay, or RNA pull-down assay. Results: The expression of XIST and ZFPM2 was increased while miR-203-3p was decreased in plasmas and MC3T3-E1 cells. Knockdown of XIST promoted the proliferation, differentiation, but limited apoptosis in MC3T3-E1 cells. . Mechanically, overexpression of XIST could reverse the bio-function of miR-203-3p transfection. Additionally, miR-203-3p inverted a series of bio-functional effects of ZFPM2. Furthermore, anti-miR-203-3p rescued si-XIST-induced downregulation of ZFPM2. Conclusion: Downregulation of lncRNA XIST promoted osteoblast proliferation and differentiation, but limited apoptosis by miR-203-3p/ZFPM2 axis.
Ossification of the posterior longitudinal ligament (OPLL) is the major cause for several deteriorate bone and joint diseases. Its development is a highly organized dynamic process as modulated by various physiological and pathophysiological factors. Both long non-coding RNAs (lncRNAs) and small non-coding RNAs (miRNAs) have been postulated to involve into almost all the biological conditions. Here, we applied high through-put transcriptome screening to unveil lncRNAs highly regulated under OPLL condition. siRNA assay in combination with western blot and quantitative PCR deciphered the lncRNA and miRNA functions in OPLL and their underlying mechanism. Here we identified an lncRNA, named Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) engaged into the development of OPLL by indirectly targeting Connexin 43 (Cx43) gene. As previously reported, Cx43 is one of the main proteins contributing to OPLL partially through enhancing inflammatory signaling. On top of that, we provided another regulatory layer that MALAT1 served as the upstream effector governing the transcription of Cx43 gene. Perturbation of MALAT1 significantly inhibited Cx43 expression, inflammation, and osteogenesis. Mechanistically, in silico analysis and experimental validation both confirmed that microRNA-1 (miR-1) was the mediator connecting MALAT1-Cx43 axis: overexpression of miR-1 diminished Cx43 expression and OPLL process; meanwhile, MALAT1 acted as miR-1 sponge to inhibit its suppressive transcription effect on downstream ossification related genes. Knock-down of MALAT1 released sequestered miR-1, which repressed Cx43 expression and associated OPLL. Likewise, induced OPLL caused by overexpression of MALAT1 can be ameliorated by enhanced miR-1 function, knock-down of Cx43 or inhibition of inflammation. More importantly, further validation using patient ligament samples from non-OPLL and OPLL individuals identified MALAT1-miR-1-Cx43 regulatory axis. Collectively, we found a novel mechanism through lncRNA-miRNA interaction that provides more insights into understanding the development of OPLL.
Signaling pathways like Wnt play a vital part in all aspects of skeletal development which include osteoblastogenesis and osteoclastogenesis. Inactivation of Wnt signaling pathway leads to bone‐related disorders, whereas activation of Wnt signaling pathway can cure bone pathologies like osteoporosis. Certain microRNA(s) have been identified that commune with Wnt signaling molecules to regulate osteogenesis. In this study we reported the identification of miR‐409‐5p as a suppressor of osteogenesis by targeting Lrp‐8 which is a positive effector of Wnt signaling. Our study showed that overexpressing miR‐409‐5p inhibits osteoblast differentiation whereas obstructing miR‐409‐5p expression by anti‐miR‐409 promotes osteoblast functions and matrix mineralization. Using tools like targetscan and 3′‐UTR luciferase reporter assay, Lrp‐8 was confirmed as a straight target of miR‐409‐5p. By over expressing miR‐409‐5p, a repression of canonical Wnt/β catenin signaling was observed. These observations were strengthened by the fact that silencing of miR‐409‐5p in ovariectomized estrogen deficient Balb/c mice restored the loss of trabecular bone microarchitecture and suppressed bone resorption. Thus, targeting miR‐409‐5p may be helpful in increasing bone density in conditions like post menopausal osteoporosis. MicroRNA (miR)‐409‐5p negatively regulates bone formation by inhibiting osteoblast differentiation. Silencing of miR‐409‐5p in ovariectomized estrogen deficient Balb/c mice restored the loss of trabecular bone microarchitecture. Thus, targeting miR‐409‐5p may be helpful in increasing bone density in conditions like post menopausal osteoporosis.
Backgrounds: Osteoporosis is a common aging-related degenerative bone disease, particularly in postmenopausal women, that have a reduced estrogen level due to aging. Various factors have been recognized to promote osteoporosis development, such as microRNAs (miRNAs), which regulate the balance between osteogenesis and osteoclasis. Aim: This study was to profile miRNAs in the ovariectomy osteoporosis mice with miRNA profiling array, and to associate the deregulated miRNAs with osteogenesis. Materials and Methods: The authors firstly profiled, with miRNAs microarray analysis, the serum miRNAs in a mouse model with postmenopausal osteoporosis, post-ovary excision. Then they validated the dysregulated miRNAs via quantitative real-time polymerase chain reaction (qRT-PCR) method. In addition, these deregulated miRNAs were subjected to a pathway analysis. Results: It was indicated by the present results that in the ovariectomized mice, ten miRNAs were upregulated, such as miR-27a-5p, miR-26a-5p, miR-106a-5p, and miR- 133a-5p and the upregulation of miR-26a-5p, miR-133a-1-5, miR-141-5p, miR-200a-5p, and miR-205-5p were validated by the followed qRT-PCR method. In addition, the pathway analysis demonstrated that these miRNAs might inhibit the bone formation via regulating osteogenesis. Conclusions: In conclusion, the authors found the upregulation of miRNAs, which downregulates osteogenesis in a mice model of postmenopausal osteoporosis. The present findings suggest the potential inhibition by miRNAs in osteogenesis in postmenopausal osteoporosis.
Objective
Chondrocyte hypertrophy, a terminal stage of chondrocyte differentiation, is essential to the endochondral bone formation and is one of the major pathological factors in osteoarthritis. This study investigated the role of microRNA‐29b (miR‐29b), which is involved in chondrogenesis, in the regulation of hypertrophy in chondrocytes.
Methods
miR‐29b expression was assessed during murine mesenchymal stem cells (mMSCs) chondrogenesis. To detect whether miR‐29b affects chondrocyte hypertrophy, the mMSCs induced toward chondrogenesis were transfected with miR‐29b or its antisense inhibitor (antagomiR‐29b). Finally, the differential effects of antagomiR‐29b on chondrocytes at different differentiation stages were evaluated by loss‐of‐function experiments.
Results
miR‐29b expression was low‐level during the early chondrogenic differentiation, however, it was changed to high level during hypertrophy. Subsequently, the gain‐of‐function and loss‐of‐function experiments had confirmed that miR‐29b promoted hypertrophy in mMSC‐derived chondrocytes. In addition, we confirmed that on day 7, when cells were treated with antagomiR‐29b, was the optimal intervention time for preventing hypertrophic phenotype of mMSCs in vitro.
Conclusion
miR‐29b regulated chondrogenesis homeostasis and enhance hypertrophic phenotype. These data suggest that miR‐29b is a key regulator of the chondrocyte phenotype derived from mMSCs and it might be a potential target for articular cartilage repair.
Various of microRNAs (miRNAs) have been reported to regulate the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), however, whether miR-134 plays a role in this biological process remains undetermined. In this present study, we firstly evaluated the chondrogenic differentiation of BMSCs by Alcian blue staining, and examined the miR-134 expression by qRT-PCR during this process. And miR-134 inhibitor was used to investigate the functions of miR-134 in chondrogenic differentiation of BMSCs by Alcian blue staining, qRT-PCR and western blot. Subsequently, the correlation between miR-134 and SMAD6 was assessed via bioinformatics analysis and dual luciferase reporter assay. Finally, the roles of SMAD6 in chondrogenic differentiation of BMSCs was also determined through Alcian blue staining, qRT-PCR and western blot. As results showed that miR-134 expression was significantly downregulated during chondrogenic differentiation, and inhibition of miR-134 obviously promoted chondrogenic differentiation. Dual luciferase reporter assay indicated that miR-134 could directly targeted the 3'-UTR of SMAD6, inhibited miR-134 expression in BMSCs could upregulate SMAD6 expression. Moreover, we found that overexpression of SMAD6 significantly promoted chondrogenic differentiation, and this SMAD6 induced promotion of chondrogenic differentiation could be reversed by miR-134 mimics. In conclusion, our findings suggested that miR-134 may act as a negative regulator during chondrogenic differentiation of BMSCs by interacting with SMAD6.
Recent studies have recognized the involvement of microRNAs (miRNAs) in the development of osteoporosis, which regulate the balance between osteogenesis and osteoclasis. In this study, we investigated the regulation by miRNA-133a-5p on the osteoblast differentiation-associated markers in the mouse osteoblast-like MC3T3-E1 cells by RUNX2. First, we manipulated the miRNA-133a level in the MC3T3-E1 cells with 20 or 40 nM miR-133a-5p mimics, miR-133a-5p inhibitor, or scramble miRNA. Then, we quantified with real-time polymerase chain reaction (qRT-PCR) the expression of Collagen I, osteocalcin (OCN), and osteopontin (OPN) in the miR-133a-5p-manipulated MC3T3-E1 cells. And the confocal microscopy was also utilized to confirm the regulation by miR-133a-5p on the expression of the three molecules. We also investigated the extracellular matrix (ECM) mineralization and the alkaline phosphatase (ALP) activity in the miR-133a-5p-manipulated MC3T3-E1 cells. In addition, we explored the possible targeting by miR-133a-5p on RUNX2, which was a well-recognized promoter to osteoblast differentiation, with luciferase reporter, qRT-PCR, and Western blotting assay. Results demonstrated that the miRNA-133a-5p mimics markedly reduced, whereas the miRNA-133a-5p inhibitor significantly promoted the expression of Collagen I, OCN, and OPN, the ECM mineralization, and the ALP activity in MC3T3-E1 cells. The alignment analysis demonstrated a high homology between miRNA-133a-5p and the 3' UTR of RUNX2. Moreover, the luciferase reporter assay demonstrated that miRNA-133a-5p targeted the 3' UTR of RUNX2, and inhibited the expression of RUNX2 in both mRNA and protein levels. In conclusion, we identified the inhibition by miRNA-133a-5p to the expression of osteoblast differentiation markers, to the ECM mineralization, and to the ALP activity in MC3T3-E1 cells, by targeting the 3' UTR of RUNX2. Our study suggests that miRNA-133a-5p might be an important target to inhibit osteoblast differentiation in osteoporosis.
Background:
Osteoarthritis (OA), as a degenerative disease, is a major problem in ageing populations. To better understand the underlying mechanisms in the pathogenesis of OA, this study was undertaken to investigate the role of microRNA (miR)-19a in chondrocytes.
Methods:
Expression of the members of miR-17-92 cluster in synovium from OA patients and non-OA patients were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). miR-19a was abnormal expressed in human chondrocyte line (CHON-001 and T-C/28 cells) and primary human chondrocytes by transient transfection. Cell viability, migration and apoptosis were determined by CCK-8 assay, wound healing assay, and flow cytometry, respectively. Expression of apoptosis related factors was measured by western blot. Transcription factor SOX9 expression and activity of NF-κB pathway were also assessed by western blot.
Results:
Levels of miR-19a and other five members of miR-17-92 cluster were down-regulated in OA patients' synovium compare with non-OA. miR-19a overexpression promoted cell viability and migration of chondrocytes, while miR-19a suppression promoted cell apoptosis, and inhibited cell viability and migration. miR-19a direct up-regulated expression of SOX9, and thus affecting cell viability and migration. miR-19a promoted activation of NF-κB signaling pathway to up-regulate SOX9 expression.
Conclusion:
miR-19a was down-regulated in synovium form OA patients. miR-19a could promote cell viability and migration of chondrocyte via positively regulating SOX9 expression through NF-κB signaling pathway. This study might provide the novel strategy for clinical treatment of OA caused by chondrocyte function degradation.
Articular cartilage enables weight bearing and near friction-free movement in the joints. Critical to its function is the
production of a specialized, mechanocompetent extracellular matrix controlled by master regulator transcription factor SOX9.
Mutations in SOX9 cause campomelic dysplasia, a haploinsufficiency disorder resulting in severe skeletal defects and dwarfism.
Although much is understood about how SOX9 regulates cartilage matrix synthesis and hence joint function, how this master
regulator is itself regulated remains largely unknown. Here we identify a specific microRNA, miR-145, as a direct regulator
of SOX9 in normal healthy human articular chondrocytes. We show that miR-145 directly represses SOX9 expression in human cells
through a unique binding site in its 3′-UTR not conserved in mice. Modulation of miR-145 induced profound changes in the human
chondrocyte phenotype. Specifically, increased miR-145 levels cause greatly reduced expression of critical cartilage extracellular
matrix genes (COL2A1 and aggrecan) and tissue-specific microRNAs (miR-675 and miR-140) and increased levels of the hypertrophic
markers RUNX2 and MMP13, characteristic of changes occurring in osteoarthritis. We propose miR-145 as an important regulator
of human chondrocyte function and a new target for cartilage repair.
Innovative strategies based on regenerative medicine, in particular tissue engineering of skeletal muscle, are promising for treatment of patients with skeletal muscle damage. However, the efficiency of satellite cell differentiation in vitro is suboptimal. MicroRNAs are involved in the regulation of cell proliferation and differentiation. We hypothesized that transient overexpression of microRNA-1 or microRNA-206 enhances the differentiation potential of human satellite cells by downregulation quiescent satellite cell regulators, thereby increasing myogenic regulator factors. To investigate this, we isolated and cultured human satellite cells from muscle biopsies. First, through immunofluorescent analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), we showed that in satellite cell cultures, low Pax7 expression is related to high MyoD expression on differentiation, and, subsequently, more extensive sarcomere formation, that is, muscle differentiation, was detected. Second, using qRT-PCR, we showed that microRNA-1 and microRNA-206 are robustly induced in differentiating satellite cells. Finally, a gain-of-function approach was used to investigate microRNA-1 and microRNA-206 potential in human satellite cells to improve differentiation potential. As a proof of concept, this was also investigated in a three-dimensional bioartificial muscle construct. After transfection with microRNA-1, the number of Pax7 expressing cells decreased compared with the microRNA-scrambled control. In differentiated satellite cell cultures transfected with either microRNA-1 or microRNA-206, the number of MyoD expressing cells increased, and α-sarcomeric actin and myosin expression increased compared with microRNA-scrambled control cultures. In addition, in a three-dimensional bioartificial muscle construct, an increase in MyoD expression occurred. Therefore, we conclude that microRNA-1 and microRNA-206 can improve human satellite cell differentiation. It represents a potential novel approach for tissue engineering of human skeletal muscle for the benefit of patients with facial paralysis.
Chondrogenic differentiation of mesenchymal stem cells (MSCs) is accurately regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process still remain to be defined. MicroRNAs (miRNAs) regulate various biological processes by binding target mRNA to attenuate protein synthesis. To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we identified that miR-145 was decreased during transforming growth factor beta 3 (TGF-β3)-induced chondrogenic differentiation of murine MSCs. Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-145 targets a putative binding site in the 3'-UTR of SRY-related high mobility group-Box gene 9 (Sox9) gene, the key transcription factor for chondrogenesis. In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels. Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.
Bone regeneration is a complex, well-orchestrated physiological process of bone formation, which can be seen during normal fracture healing, and is involved in continuous remodelling throughout adult life. However, there are complex clinical conditions in which bone regeneration is required in large quantity, such as for skeletal reconstruction of large bone defects created by trauma, infection, tumour resection and skeletal abnormalities, or cases in which the regenerative process is compromised, including avascular necrosis, atrophic non-unions and osteoporosis. Currently, there is a plethora of different strategies to augment the impaired or 'insufficient' bone-regeneration process, including the 'gold standard' autologous bone graft, free fibula vascularised graft, allograft implantation, and use of growth factors, osteoconductive scaffolds, osteoprogenitor cells and distraction osteogenesis. Improved 'local' strategies in terms of tissue engineering and gene therapy, or even 'systemic' enhancement of bone repair, are under intense investigation, in an effort to overcome the limitations of the current methods, to produce bone-graft substitutes with biomechanical properties that are as identical to normal bone as possible, to accelerate the overall regeneration process, or even to address systemic conditions, such as skeletal disorders and osteoporosis.
Our recent study showed that miR-2861 promotes osteoblast differentiation by targeting histone deacetylase 5, resulting in increased runt-related transcription factor 2 (Runx2) protein production. Here we identified another new microRNA (miRNA) (miR-3960) that played a regulatory role in osteoblast differentiation through a regulatory feedback loop with miR-2861. miR-3960 and miR-2861 were found clustered at the same loci. miR-3960 was transcribed during bone morphogenic protein 2 (BMP2)-induced osteogenesis of ST2 stromal cells. Overexpression of miR-3960 promoted BMP2-induced osteoblastogenesis. However, the inhibition of miR-3960 expression attenuated the osteoblastogenesis. Homeobox A2 (Hoxa2), a repressor of Runx2 expression, was confirmed to be a target of miR-3960. Electrophoretic mobility shift assay and chromatin immunoprecipitation experiments confirmed that Runx2 bound to the promoter of the miR-3960/miR-2861 cluster. Furthermore, overexpression of Runx2 induced miR-3960/miR-2861 transcription, and block of Runx2 expression attenuated BMP2-induced miR-3960/miR-2861 transcription. Here we report that miR-3960 and miR-2861, transcribed together from the same miRNA polycistron, both function in osteoblast differentiation through a novel Runx2/miR-3960/miR-2861 regulatory feedback loop. Our findings provide new insights into the roles of miRNAs in osteoblast differentiation.
MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.
miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.
Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellular matrix synthesis and articular cartilage degradation, mediated by several proteinases, including Adamts-5. miR-140 is one of a very limited number of noncoding microRNAs (miRNAs) specifically expressed in cartilage; however, its role in development and/or tissue maintenance is largely uncharacterized. To examine miR-140 function in tissue development and homeostasis, we generated a mouse line through a targeted deletion of miR-140. miR-140(-/-) mice manifested a mild skeletal phenotype with a short stature, although the structure of the articular joint cartilage appeared grossly normal in 1-mo-old miR-140(-/-) mice. Interestingly, miR-140(-/-) mice showed age-related OA-like changes characterized by proteoglycan loss and fibrillation of articular cartilage. Conversely, transgenic (TG) mice overexpressing miR-140 in cartilage were resistant to antigen-induced arthritis. OA-like changes in miR-140-deficient mice can be attributed, in part, to elevated Adamts-5 expression, regulated directly by miR-140. We show that miR-140 regulates cartilage development and homeostasis, and its loss contributes to the development of age-related OA-like changes.
MMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes.
Gene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors.
IGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5.
This study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.
Growing evidence shows that microRNAs (miRNAs) regulate various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; however, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast differentiation remains elusive. Here, through comprehensive analysis of miRNAs expression during osteoblast differentiation, we show that miR-206, previously viewed as a muscle-specific miRNA, is a key regulator of this process. miR-206 was expressed in osteoblasts, and its expression decreased over the course of osteoblast differentiation. Overexpression of miR-206 in osteoblasts inhibited their differentiation, and conversely, knockdown of miR-206 expression promoted osteoblast differentiation. In silico analysis and molecular experiments revealed connexin 43 (Cx43), a major gap junction protein in osteoblasts, as a target of miR-206, and restoration of Cx43 expression in miR-206-expressing osteoblasts rescued them from the inhibitory effect of miR-206 on osteoblast differentiation. Finally, transgenic mice expressing miR-206 in osteoblasts developed a low bone mass phenotype due to impaired osteoblast differentiation. Our data show that miRNA is a regulator of osteoblast differentiation.
MicroRNAs (miRNAs) interfere with translation of specific target mRNAs and are thought to thereby regulate many cellular processes. Recent studies have suggested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify a new miRNA (miR-2861) in primary mouse osteoblasts that promotes osteoblast differentiation by repressing histone deacetylase 5 (HDAC5) expression at the post-transcriptional level. miR-2861 was found to be transcribed in ST2 stromal cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-2861 enhanced BMP2-induced osteoblastogenesis, whereas inhibition of miR-2861 expression attenuated it. HDAC5, an enhancer of runt-related transcription factor 2 (Runx2) degradation, was confirmed to be a target of miR-2861. In vivo silencing of miR-2861 in mice reduced Runx2 protein expression, inhibited bone formation, and decreased bone mass. Importantly, miR-2861 was found to be conserved in humans, and a homozygous mutation in pre-miR-2861 that blocked expression of miR-2861 was shown to cause primary osteoporosis in 2 related adolescents. Consistent with the mouse data, HDAC5 levels were increased and Runx2 levels decreased in bone samples from the 2 affected individuals. Thus, our studies show that miR-2861 plays an important physiological role in osteoblast differentiation and contributes to osteoporosis via its effect on osteoblasts.
In this study, high-throughput microRNA (miRNA) expression analysis revealed that the expression of miR-140 was associated with chemosensitivity in osteosarcoma tumor xenografts. Tumor cells ectopically transfected with miR-140 were more resistant to methotrexate and 5-fluorouracil (5-FU). Overexpression of miR-140 inhibited cell proliferation in both osteosarcoma U-2 OS (wt-p53) and colon cancer HCT 116 (wt-p53) cell lines, but less so in osteosarcoma MG63 (mut-p53) and colon cancer HCT 116 (null-p53) cell lines. miR-140 induced p53 and p21 expression accompanied with G(1) and G(2) phase arrest only in cell lines containing wild type of p53. Histone deacetylase 4 (HDAC4) was confirmed to be one of the important targets of miR-140. The expression of endogenous miR-140 was significantly elevated in CD133(+hi)CD44(+hi) colon cancer stem-like cells that exhibit slow proliferating rate and chemoresistance. Blocking endogenous miR-140 by locked nucleic acid-modified anti-miR partially sensitized resistant colon cancer stem-like cells to 5-FU treatment. Taken together, our findings indicate that miR-140 is involved in the chemoresistance by reduced cell proliferation through G(1) and G(2) phase arrest mediated in part through the suppression of HDAC4. miR-140 may be a candidate target to develop novel therapeutic strategy to overcome drug resistance.
MicroRNAs (miRNA) are short non-coding RNA molecules that regulate a variety of biological processes. The role of miRNAs in BMP2-mediated biological processes is of considerable interest. A comparative miRNA array led to the isolation of several BMP2-responsive miRNAs. Among them, miR-199a* is of particular interest, because it was reported to be specifically expressed in the skeletal system. Here we demonstrate that miR-199a* is an early responsive target of BMP2: its level was dramatically reduced at 5 h, quickly increased at 24 h and remained higher thereafter in the course of BMP2-triggered chondrogenesis of a micromass culture of pluripotent C3H10T1/2 stem cells. miR-199a* significantly inhibited early chondrogenesis, as revealed by the reduced expression of early marker genes for chondrogenesis such as cartilage oligomeric matrix protein (COMP), type II collagen, and Sox9, whereas anti-miR-199a* increased the expression of these chondrogenic marker genes. A computer-based prediction algorithm led to the identification of Smad1, a well established downstream molecule of BMP-2 signaling, as a putative target of miR-199a*. The pattern of Smad1 mRNA expression exhibited the mirror opposite of miR-199a* expression following BMP-2 induction. Furthermore, miR-199a* demonstrated remarkable inhibition of both endogenous Smad1 as well as a reporter construct bearing the 3-un-translated region of Smad1 mRNA. In addition, mutation of miR-199a* binding sites in the 3'-untranslated region of Smad1 mRNA abolished miR-199a*-mediated repression of reporter gene activity. Mechanism studies revealed that miR-199a* inhibits Smad1/Smad4-mediated transactivation of target genes, and that overexpression of Smad1 completely corrects miR-199a*-mediated repression of early chondrogenesis. Taken together, miR-199a* is the first BMP2 responsive microRNA found to adversely regulate early chondrocyte differentiation via direct targeting of the Smad1 transcription factor.
Micro-RNAs (miRNAs) are important in regulating cell fate determination because many of their target mRNA transcripts are engaged in cell proliferation, differentiation, and apoptosis. DGCR8, Dicer, and Ago2 are essential factors for miRNA homeostasis. Here we show that these three factors have critical roles in osteoclast differentiation and function. Gene silencing of DGCR8, Dicer, or Ago2 by small interfering RNA revealed global inhibition of osteoclast transcription factor expression and function, decreased osteoclastogenesis, and decreased bone resorption in vitro. In vivo, CD11b(+)-cre/Dicer-null mice had mild osteopetrosis caused by decreased osteoclast number and bone resorption. These results suggest that miRNAs play important roles in differentiation and function of osteoclasts in vitro and in vivo. We found a novel mechanism mediating these results in which PU.1, miRNA-223, NFI-A, and the macrophage colony-stimulating factor receptor (M-CSFR) are closely linked through a positive feedback loop. PU.1 stimulates miRNA-223 expression, and this up-regulation is implicated in stimulating differentiation and function of osteoclasts through negative regulation of NFI-A levels. Down-regulation of NFI-A levels is important for expression of the M-CSFR, which is critical for osteoclast differentiation and function. NFI-A overexpression decreased osteoclast formation and function with down-regulation of M-CSFR levels. Forced expression of the M-CSFR in M-CSF-dependent bone marrow macrophages from Dicer-deficient mice rescued osteoclast differentiation with up-regulation of PU.1 levels. Our studies provide new molecular mechanisms controlling osteoclast differentiation and function by the miRNA system and specifically by miRNA-223, which regulates NFI-A and the M-CSFR levels.
To identify differentially expressed microRNAs (miRNAs) in human osteoarthritic (OA) cartilage and bone tissue and to determine their relevance to chondrocyte function.
Cartilage and bone was obtained from OA patients who underwent total knee joint replacement surgery or from post-mortem patients with no previous history of OA. MiRNA expression was quantified by real-time PCR (RT-PCR). Functional pathway analysis of miRNA was performed using Ingenuity Pathway Analysis. Primary chondrocytes were isolated by collagenase digestion and transfected with miRNA mimics and miRNA inhibitors using cationic lipid. Tumour Necrosis Factor-alpha (TNF-alpha) and Matrix metalloprotease 13 (MMP13) protein levels were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
In total we identified 17 miRNA that showed greater than 4-fold differential expression between OA and normal cartilage, and 30 miRNA that showed greater than 4-fold differential expression in OA bone. Functional pathway analysis of the predicted gene targets for miR-9, miR-98, which were upregulated in both OA bone and cartilage tissue, and miR-146, which was downregulated in OA cartilage, suggested that these miRNA mediate inflammatory functions and pathways. Over-expression of miR-9, miR-98 or miR-146 in isolated human chondrocytes reduced interleukin-1 beta (IL-1 beta) induced TNF-alpha production. Furthermore, inhibition and over-expression of miR-9 modulated MMP13 secretion.
We have identified a number of differentially expressed miRNAs in late-stage human OA cartilage and bone. Functional analysis of miR-9, miR-98 and miR-146 in primary chondrocytes suggests a role in mediating the IL-1 beta induced production of TNF-alpha. MiR-9, upregulated in OA tissue, was found to inhibit secretion of the collagen type II-targeting metalloproteinase MMP13 in isolated human chondrocytes.
The 21-nucleotide small temporal RNA (stRNA) let-7regulates developmental timing in Caenorhabditis elegans and probably in other bilateral animals. We present in vivo and in vitro evidence that in Drosophila melanogaster a developmentally regulated precursor RNA is cleaved by an RNA interference-like mechanism to produce mature let-7 stRNA. Targeted destruction in cultured human cells of the messenger RNA encoding the enzyme Dicer, which acts in the RNA
interference pathway, leads to accumulation of the let-7 precursor. Thus, the RNA interference and stRNA pathways intersect. Both pathways require the RNA-processing enzyme Dicer
to produce the active small-RNA component that represses gene expression.
MicroRNAs (miRNAs) are small RNAs that regulate the expression of complementary messenger RNAs. Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved. With miRNA roles identified in developmental timing, cell death, cell proliferation, haematopoiesis and patterning of the nervous system, evidence is mounting that animal miRNAs are more numerous, and their regulatory impact more pervasive, than was previously suspected.
Micro-RNAs are small non-coding RNAs that regulate target gene expression post-transcriptionally through base pairing with
the target messenger RNA. Functional characterization of micro-RNAs awaits robust experimental methods to knock-down a micro-RNA
as well as to assay its function in vivo. In addition to the recently developed method to sequester micro-RNA with 2′-O-methyl antisense oligonucleotide, we report that small interfering RNA against the loop region of a micro-RNA precursor can
be used to deplete the micro-RNA. The depletion of miR-125b by this method had a profound effect on the proliferation of adult
differentiated cancer cells, and this proliferation defect was rescued by co-transfected mature micro-RNA. This technique
has unique advantages over the 2′-O-methyl antisense oligonucleotide and can be used to determine micro-RNA function, assay micro-RNAs in vivo, and identify the contribution of a predicted micro-RNA precursor to the pool of mature micro-RNA in a given cell. miR-125b
and let-7 micro-RNAs are induced, whereas their putative targets, lin-28 and lin-41, are decreased during in vitro differentiation of Tera-2 or embryonic stem cells. Experimental increase or decrease of micro-RNA concentrations did not,
however, affect the levels of the targets, a finding that is explained by the fact that the down-regulation of the targets
appears to be mostly at the transcriptional level in these in vitro differentiation systems. Collectively these results reveal the importance of micro-RNA depletion strategies for directly
determining micro-RNA function in vivo.
Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more 'aggrecanases' from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.
MicroRNAs (miRNAs) are small noncoding RNAs, about 21 nucleotides in length, that can regulate gene expression by base-pairing
to partially complementary mRNAs. Regulation by miRNAs can play essential roles in embryonic development. We determined the
temporal and spatial expression patterns of 115 conserved vertebrate miRNAs in zebrafish embryos by microarrays and by in
situ hybridizations, using locked-nucleic acid–modified oligonucleotide probes. Most miRNAs were expressed in a highly tissue-specific
manner during segmentation and later stages, but not early in development, which suggests that their role is not in tissue
fate establishment but in differentiation or maintenance of tissue identity.
MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.
Deregulation of micro-RNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the
translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential
of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a and miR-125b within the 3′-untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR-125a or miR-125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3′ 3′-untranslated regions of ERBB2 and ERBB3 demonstrated ∼35% less activity in miR-125a- and miR-125b-expressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing
either miR-125a or miR-125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR-125a-or miR-125b-overexpressing SKBR3 cells were impaired in their anchorage-dependent growth and exhibited reduced migration and invasion
capacities. Parallel studies performed on MCF10A cells demonstrated that miR-125a or miR-125b overexpression produced only marginal influences on the growth and migration of these non-transformed human mammary epithelial
cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression
and function.
Innovative strategies based on regenerative medicine, in particular tissue engineering of skeletal muscle, are promising for treatment of patients with skeletal muscle damage. However, the efficiency of satellite cell differentiation in vitro is suboptimal. MicroRNAs are involved in the regulation of cell proliferation and differentiation. We hypothesized that transient overexpression of microRNA-1 or microRNA-206 enhances the differentiation potential of human satellite cells by downregulation quiescent satellite cell regulators, thereby increasing myogenic regulator factors. To investigate this, we isolated and cultured human satellite cells from muscle biopsies. First, through immunofluorescent analysis and quantitative reverse transcription- polymerase chain reaction (qRT-PCR), we showed that in satellite cell cultures, low Pax7 expression is related to high MyoD expression on differentiation, and, subsequently, more extensive sarcomere formation, that is, muscle differentiation, was detected. Second, using qRT-PCR, we showed that microRNA-1 and microRNA-206 are robustly induced in differentiating satellite cells. Finally, a gain-of-function approach was used to investigate microRNA-1 and microRNA-206 potential in human satellite cells to improve differentiation potential. As a proof of concept, this was also investigated in a three-dimensional bioartificial muscle construct. After transfection with microRNA-1, the number of Pax7 expressing cells decreased compared with the microRNA-scrambled control. In differentiated satellite cell cultures transfected with either microRNA-1 or microRNA-206, the number of MyoD expressing cells increased, and α-sarcomeric actin and myosin expression increased compared with microRNA-scrambled control cultures. In addition, in a three-dimensional bioartificial muscle construct, an increase in MyoD expression occurred. Therefore, we conclude that microRNA-1 and microRNA-206 can improve human satellite cell differentiation. It represents a potential novel approach for tissue engineering of human skeletal muscle for the benefit of patients with facial paralysis.
Because miR-146a is linked to osteoarthritis (OA) and cartilage degeneration is associated with pain, we have characterized the functional role of miR-146a in the regulation of human articular cartilage homeostasis and pain-related factors. Expression of miRNA 146a was analyzed in human articular cartilage and synovium, as well as in dorsal root ganglia (DRG) and spinal cord from a rat model for OA-related pain assessment. The functional effects of miR-146a on human chondrocytic, synovialm and microglia cells were studied in cells transfected with miR-146a. Using real-time PCR, we assessed the expression of chondrocyte metabolism-related genes in chondrocytes, genes for inflammatory factors in synovial cells, as well as pain-related proteins and ion channels in microglial cells. Previous studies showed that miR-146a is significantly upregulated in human peripheral knee OA joint tissues. Transfection of synthetic miR-146a significantly suppresses extracellular matrix-associated proteins (e.g., Aggrecan, MMP-13, ADAMTS-5, collagen II) in human knee joint chondrocytes and regulates inflammatory cytokines in synovial cells from human knee joints. In contrast, miR-146a is expressed at reduced levels in DRGs and dorsal horn of the spinal cords isolated from rats experiencing OA-induced pain. Exogenous supplementation of synthetic miR-146a significantly modulates inflammatory cytokines and pain-related molecules (e.g., TNFα, COX-2, iNOS, IL-6, IL8, RANTS and ion channel, TRPV1) in human glial cells. Our findings suggest that miR-146a controls knee joint homeostasis and OA-associated algesia by balancing inflammatory responses in cartilage and synovium with pain-related factors in glial cells. Hence, miR-146a may be useful for the treatment of both cartilage regeneration and pain symptoms caused by OA.
Influences of alkaline treatment on the structural properties and catalytic performances of ZSM-5/ZSM-11 composite zeolites with alumina as binder at different preparation steps were studied in the present investigation. Temperature-programmed desorption of ammonium (NH3-TPD) and pyridine infrared (Py-IR) spectra revealed that alkaline treatment sequences changed both the distribution and amount of the acidities in the ZSM-5/ZSM-11-Al2O3 samples. The mesopores created by alkaline treatment were found beneficial for the diffusion of aromatic molecules, as determined by the xylene-uptake experiments using a tapered element oscillating microbalance (TEOM). In 1-hexene isomerization and aromatization reactions, the sample after extrusion followed by alkaline treatment exhibited excellent aromatization activity and stability compared with other samples undergoing different treatment sequences. The enhanced catalytic performance could be attributed to the redistribution of acid sites and introduction of more mesopores.
MicroRNAs function as an endogenous mode of fine gene regulation and have been implicated in multiple differentiation and developmental processes. In the present study, we investigated the role of miRNA-34 during chondrogenic differentiation of chick limb mesenchymal cells. We found that the expression of miR-34a increased upon chondrogenic inhibition. Blockade of miR-34a via PNA-based antisense oligonucleotides (ASOs) recovered the chondro-inhibitory actions of JNK inhibitor on migration of chondrogenic progenitors and the formation of precartilage condensation. Furthermore, we determined that EphA5 is a relevant target of miR-34a during chondrogenesis. MiR-34a was necessary and sufficient to down-regulate EphA5 expression, and up-modulation of EphA5 is sufficient to overcome inhibitory