Article

The microRNA miR-34c inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia by targeting stem cell factor

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Abstract

The fine balance between proliferation and differentiation of vascular smooth muscle cells (VSMCs) is indispensable for the maintenance of healthy blood vessels, whereas an increase in proliferation participates in pathologic cardiovascular events such as atherosclerosis and restenosis. Here we report that microRNA-34c (miR-34c) targets stem cell factor (SCF) to inhibit VSMC proliferation and neointimal hyperplasia. In an animal model, miR-34c was significantly increased in the rat carotid artery after catheter injury. Transient transfection of miR-34c to either VSMCs or A10 cells inhibited cell survival by inducing apoptosis, which was accompanied by an increase in expression of p21, p27, and Bax. Transfection of miR-34c also attenuated VSMC migration. Bioinformatics showed that SCF is a target candidate of miR-34c. miR-34c down-regulated luciferase activity driven by a vector containing the 3'-untranslated region of SCF in a sequence-specific manner. Forced expression of SCF in A10 cells induced proliferation and migration, whereas knocking-down of SCF reduced cell survival and migration. miR-34c antagomir-induced VSMC proliferation was blocked by SCF siRNA. Delivery of miR-34c to rat carotid artery attenuated the expression of SCF and blocked neointimal hyperplasia. These results suggest that miR-34c is a new modulator of VSMC proliferation and that it inhibits neointima formation by regulating SCF. Copyright © 2015. Published by Elsevier Inc.

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... Thus, as easily assumed, various cellular functions including proliferation, apoptosis, differentiation, and migration are mediated by microRNAs. Our previous studies have demonstrated that miR-132 and miR-34c modulate VSMC phenotypic change and neointimal hyperplasia [12,13]. While miR-124 has been studied in diverse systems, its role in VSMC function and atherosclerosis remains to be investigated. ...
... Male Sprague-Dawley rats weighing approximately 250 g were anesthetized with an intraperitoneal injection of 2,2,2, tribromoethanol (300 mgÁkg À1 ). Balloon injury was induced in the carotid arteries of the rats as described previously [12,13], and arterial samples were obtained 1, 7, and 14 days later. Total RNA was extracted from the injured arterial sections with Trizol (Molecular Research Center Inc., Cincinnati, OH, USA) reagent, and the expression of micro-RNAs was observed by quantitative real-time RT-PCR. ...
... Protein samples were analyzed following the protocol described previously using antibodies at a 1 : 1000 dilution [12,13]. The image was acquired by using Luminescent Image Analyzer LAS-3000 with IMAGE READER LAS3000 software (Fujifilm Medical Systems, Stamford, CT, USA). ...
Article
S100 calcium-binding protein A4 (S100A4) induces proliferation and migration of vascular smooth muscle cells (VSMCs). We aimed to find the microRNA regulating S100A4 expression. S100A4 transcripts are abruptly increased in the acute phase of carotid arterial injury one day later (at day 1) but gradually decreases at days 7 and 14. Bioinformatics analysis reveal that miR-124 targets S100A4. VSMC survival is attenuated by miR-124 mimic but increased by miR-124 inhibitor. miR-124 decreases immediately after carotid arterial injury but dramatically increases at days 7 and 14. miR-124 inhibitor-induced cell proliferation is blocked by S100A4 siRNA, whereas miR-124-induced cell death is recovered by S100A4. Our findings suggest that miR-124 is a novel regulator of VSMC proliferation and may play a role in the development of neointimal proliferation. This article is protected by copyright. All rights reserved.
... Среди штаммов НEV, причастных к этим заболеваниям, группа вирусов Коксаки В (CVB) изучена более детально. Были предложены несколько механизмов, с помощью которых CVB может инициировать или ускорить аутоиммунные поцессы, в том числе -прямая индукция апоптоза β-клеток, содействие продукции провоспалительных цитокинов, активация TLR, молекулярная мимикрия (активация аутореактивных Т-и В-клеток перекрестно-реагирующими эпитопами инфекционного возбудителя), а также нарушение регуляции микроРНК хозяина [Kim et al. 2015]. CVB5-инфекция может вызвать в островках поджелудочной железы человека активацию многих генов иммунного ответа, в том числе -PRR (pattern recognition receptors). ...
... Так, уровни мРНК рецептора группы RIG-I-подобных рецепторов IFIH1 (MDA5 -Melanoma Differentiation-Associated protein) и TLR7 были значительно повышены в CVB5-инфицированных клетках по сравнению с контрольными. Исследование других генов, связанных с риском СД1, продемонстрировало увеличение количества мРНК генов адаптерного белка SH2B3 (SH2B adaptor protein) и IKZF1(Ikaros family zinc finger protein 1) [Kim et al. 2015]. ...
... МикроРНК-34c была идентифицирована как новый модулятор, который ингибирует деление ГМК и рост неоинтимы, действуя на SCF. МикроРНК-34c уменьшает фосфорилирование ERK и увеличивает экспрессию KLF4, p21Cip1, p27Kip1 и Bax, что предполагает участие в регуляции ГМК сигнального пути SCF/ERK [Choe et al. 2015 МикроРНК-133 может усиливать экспрессию IGF-1R, продлевая период полураспада его мРНК. У мышей ApoE-/-ГМК из поздних атеросклеротических поражений характеризуются более низкими уровнями экспрессии микроРНК-133a/IGF-1R и ослаблением IGF-1-стимулированной пролиферации. ...
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В книге обобщены новые данные по биохимическим и сигнальным механизмам, лежащим в основе патогенеза диабета и ряда его осложнений. Описаны механизмы, участвующие в рецепции и проведении сигналов инсулина в клетках-мишениях. Значительное внимание уделяется роли хронического воспаления и ключевого фактора воспаления NF-κB в патогенезе обоих типов диабета. Рассматривается связь диабета с атеросклерозом, когнитивными нарушениями, злокачественной трансформацией клеток. Отмечена роль эпигенетических факторов в развитии диабета и атеросклероза. Описаны также механизмы действия метформина и других сахароснижающих препаратов, используемых для лечения диабета. Книга будет полезна эндокринологам, медикам, биохимикам и молекулярным биологам.
... Previous studies have identified a considerable number of molecular players with critical roles in the proliferation of VSMCs [14]. Noncoding RNAs (ncRNAs), such as long (> 200nt) ncRNAs (lncRNAs) and miRNAs, were reported to be involved in regulating various behaviors of VSMCs [15,16]. For instance, LncRNA BRAF-activated noncoding RNA (BANCR) interacted with the JNK pathway to promote the proliferation of VSMCs [15]. ...
... For instance, LncRNA BRAF-activated noncoding RNA (BANCR) interacted with the JNK pathway to promote the proliferation of VSMCs [15]. MiR-34c suppressed the proliferation of VSMCs by downregulating stem cell factor [16]. LncRNA KCNQ1OT1 facilitated osteosarcoma cell proliferation via competitively binding miR-34c [17]. ...
... Although miR-34c is considered to be a key regulator of VSMC proliferation, it is not clear whether it is involved in atherosclerosis [16,18]. To the best of our knowledge, this study is the first time to report that miR-34c downregulated in atherosclerosis. ...
Article
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Aberrant vascular smooth muscle cell (VSMCs) proliferation involves in the development of atherosclerosis. It reported that Long noncoding BRAF-activated noncoding RNA (BANCR) and miR-34c played opposite roles in the regulation of the proliferation of VSMCs, indicating that there might be a potential interaction between them. This study was to investigate the relationship between BANCR and miR-34c in atherosclerosis. Blood (5 ml) was obtained from 56 patients with atherosclerosis and 56 healthy volunteers after they were fasted overnight, and plasma was extracted from the blood. Human Aortic Smooth Muscle Cells (HASMCs) were used to perform in vitro cell experiments. RT-qPCR was performed to measure the expression of BANCR and miR-34c in plasma and HASMCs. Dual luciferase reporter assay detected the interaction between BANCR and miR-34c. CCK-8 assay was used to assess the effects of BANCR and miR-34c overexpression on the proliferation of HASMCs. Western blotting was used to assess the effects of BANCR and miR-34c overexpression on the protein expression of HMGB1, TNF-ɑ and Bcl-2. In this study, we found that BANCR was upregulated, while miR-34c was downregulated in atherosclerosis. Bioinformatics analysis showed that BANCR and miR-34c could directly interact with each other. Moreover, overexpression of BANCR could decrease the expression of miR-34c in HASMCs, but overexpression of miR-34c could not affect the expression of BANCR. Furthermore, overexpression of BACNR increased miR-34c methylation, and knockdown of endogenous BANCR decreased miR-34c methylation. In addition, overexpression of BANCR reduced the effects of miR-34c on HASMCs proliferation and reversed the effects of miR-34c on HMGB1, TNF-ɑ and Bcl-2 expression. BANCR overexpression could induce HASMCs proliferation by downregulating the miR-34c methylation. Therefore given BANCR upregulation in atherosclerosis, its expression may be considered as a novel and useful biomarker for atherosclerosis prevention and prognosis. However considering the possible effects of other underlying diseases on both BANCR expression and miR-34c in atherosclerosis, further investigation is suggested for future research.
... Two members of the miR-34 family, miR-34a and miR-34c, have both been demonstrated to play protective roles in injury-induced neointimal formation [123,124]. VSMC proliferation and migration were perturbed by over-expression of miR-34a and miR-34c, through direct regulation of Notch1 [123] and SCF [124] respectively. Of note, despite multiple lines of evidence showing a role for miR-34a in the regulation of endothelial cell behaviour, re-endothelialisation was not assessed in the aforementioned studies, which is of concern given that a wire-injury endothelial denudation model was used [123], and aberrant neointimal formation is attributed in part to delayed re-endothelialisation [125]. ...
... Two members of the miR-34 family, miR-34a and miR-34c, have both been demonstrated to play protective roles in injury-induced neointimal formation [123,124]. VSMC proliferation and migration were perturbed by over-expression of miR-34a and miR-34c, through direct regulation of Notch1 [123] and SCF [124] respectively. Of note, despite multiple lines of evidence showing a role for miR-34a in the regulation of endothelial cell behaviour, re-endothelialisation was not assessed in the aforementioned studies, which is of concern given that a wire-injury endothelial denudation model was used [123], and aberrant neointimal formation is attributed in part to delayed re-endothelialisation [125]. ...
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Cardiovascular diseases encompassing atherosclerosis, aortic aneurysms, restenosis, and pulmonary arterial hypertension, remain the leading cause of morbidity and mortality worldwide. In response to a range of stimuli, the dynamic interplay between biochemical and biomechanical mechanisms affect the behaviour and function of multiple cell types, driving the development and progression of cardiovascular diseases. Accumulating evidence has highlighted microRNAs (miRs) as significant regulators and micro-managers of key cellular and molecular pathophysiological processes involved in predominant cardiovascular diseases, including cell mitosis, motility and viability, lipid metabolism, generation of inflammatory mediators, and dysregulated proteolysis. Human pathological and clinical studies have aimed to identify select microRNA which may serve as biomarkers of disease and their progression, which are discussed within this review. In addition, I provide comprehensive coverage of in vivo investigations elucidating the modulation of distinct microRNA on the pathophysiology of atherosclerosis, abdominal aortic aneurysms, restenosis, and pulmonary arterial hypertension. Collectively, clinical and animal studies have begun to unravel the complex and often diverse effects microRNAs and their targets impart during the development of cardiovascular diseases and revealed promising therapeutic strategies through which modulation of microRNA function may be applied clinically.
... However, the mechanism of SCF/c-kit signaling pathway on ICCs remains unknown. SCF was confirmed as a target of miR-34c-5p in colorectal cancer and vascular smooth muscle cells (Choe et al. 2015;Yang et al. 2014). In the present study, we found decreased number of ICCs accompanied with the activation of M1 macrophage in STC model rats. ...
... Bioinformatics analysis and luciferase reporter assay revealed that SCF is a direct target of miR-34c-5p. SCF was also confirmed as a target of miR-34c-5p in colorectal cancer and vascular smooth muscle cells (Choe et al. 2015;Yang et al. 2014). In vivo studies showed that STC rats treated with exosomes from miR-34c-5p overexpression macrophages had a severe decrease of ICCs, which could be reversed by treatment with exosomes from miR-34c-5p downregulated macrophages. ...
Article
Slow transit constipation (STC) is a gastrointestinal disorder characterized by abnormal prolonged colonic transit time, which affects the life quality of many people. The decrease number of interstitial cells of Cajal (ICCs) is involved in the pathogenesis of STC. However, the molecular mechanism of loss of ICCs in STC remains unclear, making it difficult to develop new agents for the disease. In this study, we investigated the mechanism of decreasing ICCs in the pathogenesis of STC. We constructed the STC model rats by using atropine and diphenoxylate. A series of methods were used including immunofluorescence and immunochemistry staining, western blot, qRT-PCR, exosomes extraction and exosomes labeling. The results indicate that ICCs decreased in the STC rats accompanied with the macrophages activation. Further studies suggested that macrophages decreased the cell viability of ICCs by secretion exosomes containing miR-34c-5p. miR-34c-5p targeted the 3′-UTR of stem cell factor(SCF) mRNA and regulated the expression of SCF negatively. In conclusion, we demonstrated a novel regulatory mechanism of ICCs cell viability in STC. We found that exosome miR-34c-5p mediate macrophage-ICCs cross-talk. M1 macrophages derived exosomes miR-34c-5p decreased ICCs cell viability by directly targeting SCF.
... However, with the progress of aging and in response to various pathological stimuli, VSMCs deviate from their physiological state and switch to a proliferative, migratory, apoptotic, and differentiation phenotype, which is called phenotypic modulation or switching [73]. Recently, emerging evidence has revealed that miRNAs are involved in vascular disease through the regulation of VSMC migration, proliferation, differentiation, and apoptosis [74][75][76][77]. Next, we will summarize the current knowledge on the role of miRNAs in the regulation of VSMCs functions, including proliferation, migration, apoptosis, and differentiation ( Figure 2). ...
... As mentioned in the miRNAs participate in the function of ECs, miR-34a and miR-34c inhibited VSMCs proliferation and migration through the modulation of Notch gene and stem cell factor (SCF) expression, respectively, which contributed to reducing neointimal hyperplasia [75,112]. Specifically, overexpression of miR-223 and miR-153 inhibited stretch stress-enhanced VSMCs proliferation via activation of the insulin-like growth factor-1 receptor and PI3K-AKT signaling pathway [113]. ...
Article
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Vascular aging, a specific type of organic aging, is related to age-dependent changes in the vasculature, including atherosclerotic plaques, arterial stiffness, fibrosis, and increased intimal thickening. Vascular aging could influence the threshold, process, and severity of various cardiovascular diseases, thus making it one of the most important risk factors in the high mortality of cardiovascular diseases. As endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are the main cell biological basis of these pathology changes of the vasculature, the structure and function of ECs and VSMCs play a key role in vascular aging. MicroRNAs (miRNAs), small noncoding RNAs, have been shown to regulate the expression of multiple messenger RNAs (mRNAs) posttranscriptionally, contributing to many crucial aspects of cell biology. Recently, miRNAs with functions associated with aging or aging-related diseases have been studied. In this review, we will summarize the reported role of miRNAs in the process of vascular aging with special emphasis on EC and VSMC functions. In addition, the potential application of miRNAs to clinical practice for the diagnosis and treatment of cardiovascular diseases will also be discussed.
... Though numerous studies had demonstrated that miRNAs played an important role in regulating vascular calcification [20,21], the specific mechanisms involved still needed to be elucidated. Another important miRNA, miR-34b, also played important roles in skeletal and muscle development, physiology, and disease pathogenesis [22,23]. For example, Wei et al. found that miR-34b inhibited osteoblast proliferation and differentiation in mice by targeting Satb2 [9]. ...
... Hu et al. showed that miR-1298 was regulated by DNA methylation and affected the proliferation and migration of VSMCs by targeting connexin 43 [14]. Another study showed that miR-143 was hypermethylated, which was regulated by DNMT3a in Hcy-induced proliferation of VSMCs [22,25]. Our previous study also demonstrated that the methylation level of miR-204 was regulated by DNMT3a in the process of arterial calcification [26]. ...
Article
Vascular calcification is one of the most important factors for cardiovascular and all-cause mortality in patients with end-stage renal diseases (ESRD). The current study was aimed to investigate the function and mechanisms of miR-34b on the calcification of vascular smooth muscle cells (VSMCs) both in vitro and in vivo. We found that the expression of miR-34b was significantly suppressed in VSMCs with high inorganic phosphate (Pi) treatment, as well as mouse arteries derived from 5/6 nephrectomy with a high-phosphate diet (0.9% Pi, 5/6 NTP) and human renal arteries from uraemia patients. Overexpression of miR-34b alleviated calcification of VSMCs, while VSMCs calcification was enhanced by inhibiting the expression of miR-34b. Bisulphite sequencing PCR (BSP) uncovered that CpG sites upstream of miR-34b DNA were hypermethylated in calcified VSMCs and calcified arteries due to 5/6 NTP, as well as calcified renal arterial tissues from uraemia patients. Meantime, increased DNA methyltransferase 3a (DNMT3a) resulted in the hypermethylation of miR-34b in VSMCs, while 5-aza-2'-deoxycytidine (5-aza) reduced the methylation rate of miR-34b and restored the expression of miR-34b in VSMCs. When DNMT3a was knocked down using DNMT3a siRNA, the effect of 3.5 mM of Pi on calcification of VSMCs was abrogated. In addition, Notch1 was validated as the functional target of miR-34b and involved in the process of calcification of VSMCs. Taken together, our data showed a specific role for miR-34b in regulating calcification of VSMCs both in vitro and in vivo, which was regulated by upstream DNA methylation of miR-34b and modulated by the downstream target gene expression, Notch1. These results suggested that modulation of miR-34b may offer new insight into a novel therapeutic approach for vascular calcification.
... Results showed that members of the miR-34c family (miR34c-5p, miR34c-3p, and miR34b-3p) were most highly expressed, followed by miR-132-5p/ 3p, miR-381-3p, and miR-409-5p in the aorta of angiotensin II-treated mice. Studies on miR-34c, miR-132-5p, and miR-132-3p have been previously published [19][20][21][22]. It has also been reported that miR-212 expression is increased or decreased in various cancers, suggesting its potential role as a biomarker [23]. ...
... In accordance with previous findings, our microarray results also showed that miR-34c-5p and miR-34c-3p expression was most highly induced in response to angiotensin II treatment [27,28]. Moreover, miR-34c and miR-34a have been reported to inhibit VSMC proliferation by targeting stem cell factor and neurogenic locus notch homolog protein-1, respectively [19,29]. Hence, we further investigated the role of miR-212-5p in this study. ...
Article
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Vascular remodeling and contraction contribute to the development of hypertension. We investigated the role of miR-212-5p and its downstream target in vascular smooth muscle cell (VSMC) proliferation, migration, and contraction. MicroRNA microarray and PCR analyses showed that miR-212-5p expression was increased with angiotensin II treatment in vivo and in vitro. Moreover, miR-212-5p mimic treatment attenuated and miR-212-5p inhibitor treatment increased VSMC proliferation and migration. Additionally, miR-212-5p mimic treatment suppressed VSMC contraction and related gene expression [Ras homolog gene family member A (RhoA) and Rho-associated protein kinase 2], while miR-212-5p inhibitor treatment exerted opposite effects. Bioinformatics analysis revealed that platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is a target of miR-212-5p. miR-212-5p mimic treatment significantly reduced and miR-212-5p inhibitor treatment increased PAFAH1B2 expression. Furthermore, PAFAH1B2 expression was decreased in angiotensin II-treated aortic tissues and VSMCs. PAFAH1B2 was ubiquitously expressed in most adult rat tissues. In the vasculature, PAFAH1B2 was only distributed in the cytoplasm. PAFAH1B2 overexpression decreased A10 cell proliferation, while PAFAH1B2 knockdown increased A10 cell proliferation and cyclin D1 mRNA levels. PAFAH1B2 knockdown stimulated VSMC contraction and RhoA expression. These results suggest that miR-212-5p and PAFAH1B2 are novel negative regulators of VSMC proliferation, migration, and contraction in hypertension.
... Stem cell factor (SCF) [35] miRNA-124 ...
... It was demonstrated that the overexpression of miRNA-34a in serum-starved VSMCs significantly inhibited VSMC proliferation and migration, while knockdown of miRNA-34a dramatically promoted VSMC proliferation by targeting neurogenic locus notch homolog protein-1 (Notch1), a single-pass transmembrane receptor [34]. The miRNA-34c inhibited VSMC proliferation and neointimal hyperplasia by targeting the stem cell factor (SCF), a cytokine that binds to the c-kit receptor [35]. miRNA-141 inhibited oxLDL-induced abnormal VSMC proliferation through targeting pregnancy-associated plasma protein A (PAPP-A), a secreted protease targeting IGF-1 binding proteins [40]. ...
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Vascular smooth muscle cell (VSMC) proliferation plays a critical role in atherosclerosis. At the beginning of the pathologic process of atherosclerosis, irregular VSMC proliferation promotes plaque formation, but in advanced plaques VSMCs are beneficial, promoting the stability and preventing rupture of the fibrous cap. Recent studies have demonstrated that microRNAs (miRNAs) expressed in the vascular system are involved in the control of VSMC proliferation. This review summarizes recent findings on the miRNAs in the regulation of VSMC proliferation, including miRNAs that exhibit the inhibition or promotion of VSMC proliferation, and their targets mediating the regulation of VSMC proliferation. Up to now, most of the studies were performed only in cultured VSMC. While the modulation of miRNAs is emerging as a promising strategy for the regulation of VSMC proliferation, most of the effects of miRNAs and their targets in vivo require further investigation.
... miR-34c, a member of miR-34 family, involved in the regulation of various biological processes, such as spermatogenesis (Bouhallier et al. 2010), male infertility (Wang et al. 2011;Yu et al. 2014), and oncogene/tumour suppressor networks (Cannell and Bushell 2010). It was identified to be a pro-apoptotic and anti-proliferative factor in many cell types, including vascular smooth muscle cell (Choe et al. 2015) and various cancer cells (Corney et al. 2007;Cai et al. 2010;Hagman et al. 2010;Li et al. 2015). However, the roles of miR-34c in GCs function remain unknown. ...
... ** , P<0.01. (Choe et al. 2015), nasopharyngeal carcinoma cells (Li et al. 2015), neoplastic cells (Corney et al. 2007), laryngeal carcinoma cells (Cai et al. 2010), and prostate cancer cells (Hagman et al. 2010). However, the roles of miR-34c in GCs function remain unknown. ...
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Granulosa cells (GCs) are somatic cells of ovary, the behaviors of GCs are important for ovarian function. MicroRNAs (miRNAs) are a class of endogenous 18–24 nucleotide (nt) non-coding RNAs, some of which have been shown to be important regulators of GCs function. miR-34c involved in the regulation of various biological processes and was identified to be a pro-apoptotic and anti-proliferative factor in many cell types. However, the roles of miR-34c in GCs function remain unknown. In this study, we used Annexin V-FITC and EdU assays to demonstrate that miR-34c exerted pro-apoptotic and anti-proliferative effects in porcine GCs. Dual-luciferase reporter assays, quantitative real-time PCR (qRT-PCR) and Western blotting identified Forkhead box O3a (FoxO3a) as a direct target gene of miR-34c. The overexpression of FoxO3a rescued the phenotypic change caused by miR-34c in porcine GCs. In conclusion, miR-34c regulate the function of porcine GCs by targeting FoxO3a.
... A previous study indicated miR-34a inhibits mouse smooth muscle cell proliferation by directly targeting Notch1 26 . Another miR-34 family member miR-34c also has been shown to inhibit rat vascular smooth muscle cell proliferation 27 . But, the role of miR-34 plays in pig skeletal muscle development has not been reported. ...
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Since pork accounts for about 40% of global meat consumption, the pig is an important economic animal for meat production. Pig is also a useful medical model for humans due to its similarity in size and physiology. Understanding the mechanism of muscle development has great implication for animal breeding and human health. Previous studies showed porcine muscle satellite cells (PSCs) are important for postnatal skeletal muscle growth, and Notch1 signaling pathway and miRNAs regulate the skeletal muscle development. Notch1 signal pathway regulates the transcription of certain types of miRNAs which further affects target gene expression. However, the specific relationship between Notch1 and miRNAs during muscle development has not been established. We found miR-34c is decreased in PSCs overexpressed N1ICD. Through the overexpression and inhibition of mi-34c, we demonstrated that miR-34c inhibits PSCs proliferation and promotes PSCs differentiation. Using dual-luciferase reporter assay and Chromatin immunoprecipitation, we demonstrate there is a reciprocal regulatory loop between Notch1 and miR-34c. Furthermore, injection of miR-34c lentivirus into mice caused repression of gastrocnemius muscle development. In summary, our data revealed that miR-34c can form a regulatory loop with Notch1 to repress muscle development, and this result expands our understanding of muscle development mechanism.
... ful for HUVEC to survive under hypoxia. It is unlikely that the increase from the CCK8 assay was a result of increased proliferation, as miR-34c suppresses the proliferation of vascular smooth muscle cells (Choe et al., 2015). ...
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Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.
... МикроРНК-34c был идентифицирован как новый модулятор, который ингибирует деление ГМК и неоинтиму, действуя на SCF. МикроРНК-34c уменьшает фосфорилирование ERK и увеличивает экспрессию KLF4, p21, p27 и Bax, что предполагает участие в регуляции ГМК сигнального пути SCF/ERK [9]. ...
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The review summarizes and analyzes epigenetic changes accompanying cardiovascular complications in diabetes. Data on the participation of epigenetic modifications in pathological changes of endothelial cells, smooth muscle cells and macrophages leading to atherosclerosis are presented. The role of various miRNAs in the differentiation, activa- tion, inflammation, proliferation and migration of vascular cells is described. It has been shown that histone modifica- tions, DNA methylation and miRNA spectrum change participate in the initiation and development of cardiovascular diseases in diabetes, and their study and application of the acquired data has great diagnostic, prognostic, and thera- peutic potential. Key words: atherosclerosis, diabetes, epigenetic modifications, miRNA.
... МикроРНК-34c был идентифицирован как новый модулятор, который ингибирует деление ГМК и неоинтиму, действуя на SCF. МикроРНК-34c уменьшает фосфорилирование ERK и увеличивает экспрессию KLF4, p21, p27 и Bax, что предполагает участие в регуляции ГМК сигнального пути SCF/ERK [9]. ...
Preprint
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The review of the literature summarizes and analyzes the material devoted to epigenetic changes accompanying cardiovascular complications in diabetes. Data on the participation of epigenetic modifications in pathological changes in endothelial cells, smooth muscle cells and macrophages leading to atherosclerosis are presented. The role of various miRNAs in the differentiation, activation, inflammation, proliferation and migration of vascular cells is described. It has been shown that histone modifications, DNA methylation and miRNA spectrum change participate in the initiation and development of cardiovascular diseases in diabetes, and their study and application of the acquired data has great diagnostic, prognostic, and in the long term therapeutic potential. Key words: atherosclerosis, diabetes, epigenetic modifications, miRNA.
... Many experimental results show that miRNAs affect the function of VSMCs through targeting certain genes. Among them, microRNA-34c targets stem cell factor to inhibit VSMC proliferation and neointimal hyperplasia (Choe et al., 2015); the microRNA-143/-145 cluster is essential for differentiation of VSMCs and determines VSMC phenotypic switching ; miR-181b enhances the proliferation and migration of VSMCs through activation of PI3K and MAPK pathways ; miR-146b-5p is necessary for PDGFinduced VSMC phenotype transition ; miR-638 is a key molecule in regulating human VSMC proliferation and migration by targeting the NOR1/cyclin D pathway (Li et al., 2013a). ...
Article
MiR-206 has been found to play a critical role in skeletal muscle proliferation, differentiation and regeneration. However, little is known about the function of miR-206 in vascular smooth muscle cells (VSMCs) biology. In this study, we will investigate its roles in phenotypic switching of VSMCs and neointimal lesion formation. First, we identified the expression of miR-206 in VSMCs treated with various concentrations of TGFβ1 and in rat carotid arteries after angioplasty by using qPCR. TGFβ1 inhibited the expression of miR-206 and TGFβ1 inhibitor induced miR-206 expression. In VSMCs of injured vascular walls, miR-206 expression was upregulated. Then, we overexpressed miR-206 using lentivirus Lv-rno-mir-206 and knocked down miR-206 using LV-rno-mir-206-inhibitor in rat carotid arteries after angioplasty. Overexpression of miR-206 resulted in decreasing SM22α expression in VSMCs in vitro and knockdown of miR-206 suppressed neointimal lesion formation in vivo. Finally, ZFP580 (zinc finger protein 580) was identified as the direct target of miR-206 in VSMCs by using luciferase report assay. The results indicate that miR-206 is involved in phenotypic switching of VSMCs and neointimal lesion formation after angioplasty through targeting ZFP580. These findings may provide a novel therapeutic target in post-angioplasty restenosis.
... Local miR-29b delivery decreased IH formation possibly via downregulation of MMP-2 and myeloid leukemia cell differentiation MCL-1, a known inhibitor of apoptosis [83]. Similarly, delivery of miR-34c to the rat carotid artery after catheter injury has shown to attenuate stem cell factor expression and subsequently mitigated formation of IH [84]. MiR-132 delivery also mitigated IH via inhibition of VSMC proliferation in a carotid artery injury model [85]. ...
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Background Cardiovascular disease remains a major health care challenge. The knowledge about the underlying mechanisms of the respective vascular disease etiologies has greatly expanded over the last decades. This includes the contribution of microRNAs, endogenous non-coding RNA molecules, known to vastly influence gene expression. In addition, short interference RNA has been established as a mechanism to temporarily affect gene expression. This review discusses challenges relating to the design of a RNA interference therapy strategy for the modulation of vascular disease. Despite advances in medical and surgical therapies, atherosclerosis (ATH), aortic aneurysms (AA) are still associated with high morbidity and mortality. In addition, intimal hyperplasia (IH) remains a leading cause of late vein and prosthetic bypass graft failure. Pathomechanisms of all three entities include activation of endothelial cells (EC) and dedifferentiation of vascular smooth muscle cells (VSMC). RNA interference represents a promising technology that may be utilized to silence genes contributing to ATH, AA or IH. Successful RNAi delivery to the vessel wall faces multiple obstacles. These include the challenge of cell specific, targeted delivery of RNAi, anatomical barriers such as basal membrane, elastic laminae in arterial walls, multiple layers of VSMC, as well as adventitial tissues. Another major decision point is the route of delivery and potential methods of transfection. A plethora of transfection reagents and adjuncts have been described with varying efficacies and side effects. Timing and duration of RNAi therapy as well as target gene choice are further relevant aspects that need to be addressed in a temporo-spatial fashion. Conclusions While multiple preclinical studies reported encouraging results of RNAi delivery to the vascular wall, it remains to be seen if a single target can be sufficient to the achieve clinically desirable changes in the injured vascular wall in humans. It might be necessary to achieve simultaneous and/or sequential silencing of multiple, synergistically acting target genes. Some advances in cell specific RNAi delivery have been made, but a reliable vascular cell specific transfection strategy is still missing. Also, off-target effects of RNAi and unwanted effects of transfection agents on gene expression are challenges to be addressed. Close collaborative efforts between clinicians, geneticists, biologists, and chemical and medical engineers will be needed to provide tailored therapeutics for the various types of vascular diseases.
... Significantly, the introduction of miR-34c specific inhibitor abolished the stretch-induced SCF decrease, again, suggesting that the highly expressed miR-34c in the bowel SMCs under the stretched condition was one of the substantial mediators on SCF production. In addition, the regulation of miR-34c on SCF was recently reported in human vascular SMCs and colorectal cancer cells [26,27], further supporting our results. ...
Article
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Gastrointestinal motility disorders (GMDs) are attributed to loss of interstitial cells of Cajal (ICC), whose survival and function are deeply dependent on the activation of KIT/SCF signalling. Based on the facts that gastrointestinal distention is common in GMD patients and SCF produced by smooth muscle cells (SMCs) is usually decreased before ICC loss, we considered a possible contribution of persistent gastrointestinal distention/stretch to SCF deficiency. In this study, chronic colonic distention mouse model, diabetic gastrointestinal paresis mouse model, cultured mouse colonic SMCs and colon specimens from Hirschsprung's disease patients were used. The results showed that SCF was clearly decreased in distent colon of mice and patients, and microRNA array and real-time PCR indicated a concomitant increase of miR-34c in distent colon. A negative regulation of miR-34c on SCF expression was confirmed by luciferase reporter assays together with knock-down and overexpression of miR-34c in cultured colonic SMCs. Using EMSA and ChIP assays, we further consolidated that in response to persistent stretch, the transcription factor AP-1/c-Jun was highly activated in colonic SMCs and significantly promoted miR-34c transcription by binding to miR-34c promoter. Knock-down or overexpression of AP-1/c-Jun in cultured colonic SMCs leads to down- or up-regulation of miR-34c, respectively. In addition, the activation of AP-1/c-Jun was through ERK1/2 signalling provoked by Ca(2+) overload in colonic SMCs that were subject to persistent stretch. In conclusion, our data demonstrated that persistent distention/stretch on colonic SMCs could suppress SCF production probably through Ca(2+) -ERK-AP-1-miR-34c deregulation, resulting in ICC loss or impairment and GMD progress.
... Several microRNAs have been identified that participate in VSMC differentiation, such as miR-145/143 [9], miR-221/222 [10], miR-200c [11], miR-146a [12], miR-34 [13], and mir-130a [14]. Recent studies have revealed that FOXP3induced miR-146a/b prevented tumor cell proliferation and enhanced apoptosis [15]. ...
Article
Vascular smooth muscle cells (VSMCs) play pivotal roles in the development of vascular diseases. While microRNAs are important in vascular pathologies, a few is known about their functional roles in VSMC phenotypes. We profiled microRNA expression in PDGF-BB treated VSMCs and found microRNA-146b-5p (miR-146b-5p) was upregulated. Inhibition of miR-146b-5p blocked in response to PDGF while reducing VSMC proliferation and migration. These studies implicate miR-146b-5p as necessary for PDGF-induced VSMC phenotype transition. Downstream miR-146b-5p targets modulating VSMC phenotypes will be further identified. Our study will help to understand the role of VSMCs in the pathology of vascular diseases.
... In some diseases, such as different types of cancer and hepatitis C, the therapeutic potential of miRNAs is already being tested in terms of clinical trials [38][39][40][41]. As for cardiovascular disease, [42][43][44][45]. In this regard, the local application of miRNA or anti-miRNA in the vascular wall is a feasible and promising strategy in clinical cardiology based on the possibility to create -miRNA-eluting-stents‖ to increase the re-endothelialization and decrease proliferation of the VSMC. ...
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MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Through specific base pairing with their targets messenger RNAs (mRNA), miRNA can modify cell phenotype and function. Several miRNAs are aberrantly expressed in diseased arteries and may influence different features of vascular remodeling, including neointimal formation and diminished re-endothelialization. This review will discuss the clinical implications of miRNAs in the field of vascular remodeling and their potential role as diagnostic and therapeutic tools. miRNA modulation offers a promising strategy for therapeutic intervention to inhibit smooth muscle cell proliferation and enhance endothelial regeneration after percutaneous coronary intervention (PCI) in order to reduce restenosis and late thrombosis.
... Recent research indicates that miR-34c, acting as an important tumor suppressor, can inhibit proliferation of many kinds of cancer cell proliferation. [35][36][37][38][39] Previous studies have demonstrated that the miR-34 family can directly target cell cycle genes such as CCND1, CCNE2, CDK4, and CDK6. [40][41][42][43][44] These target genes are important for cell proliferation, overexpression of miR-34c induced cell cycle arrest in G0/G1 stage and reduced the number of EdU C cells. ...
Article
miRNAs are increasingly being implicated as key regulators of cell proliferation, apoptosis, and differentiation. miRNA-34c appears to play a crucial role in cancer pathogenesis wherein it exerts its effect as a tumor suppressor. However, the role of miR-34c in myoblast proliferation remains poorly understood. Here, we found that overexpression miR-34c inhibited myoblasts proliferation by reducing the protein and mRNA expression of cell cycle genes. In contrast, blocking the function of miR-34c promoted myoblasts proliferation and increased the protein and mRNA expression of cell cycle genes. Moreover, miR-34c directly targeted YY1 and inhibited its expression. Similar to overexpression miR-34c, knockdown of YY1 by siRNA suppressed myoblasts proliferation. Our study provides novel evidence for a role of miR-34c in inhibiting myoblasts proliferation by repressing YY1. Thus, miR-34c has the potential to be used to enhance skeletal muscle development and regeneration.
... The stem cells within the endometrium have a very limited population thus it needs to have focused some attempts to establish an in vitro culture condition to expand the number of stem cells (1). Stem cell factor (SCF) is a transcriptional factor which plays crucial roles in normal proliferation, differentiation and survival in a range of cell types (15,16). Binding of SCF to its receptor c-Kit can 4 1 T trigger several4 1 T intracellular signaling 4 1 T pathways4 1 T (17)(18)(19)(20). ...
Article
Background: Stem cell factor (SCF) is a transcriptional factor which plays crucial roles in normal proliferation, differentiation and survival in a range of stem cells. Objective: The aim of the present study was to examine the proliferation effect of different concentrations of SCF on expansion of human endometrial CD146(+) cells. Materials and methods: In this experimental study, total populations of isolated human endometrial suspensions after fourth passage were isolated by magnetic activated cell sorting (MACS) into CD146(+) cells. Human endometrial CD146(+) cells were karyotyped and tested for the effect of SCF on proliferation of CD146(+) cells, then different concentrations of 0, 12.5, 25, 50 and 100 ng/ml was carried out and mitogens-stimulated endometrial CD146(+) cells proliferation was assessed by MTT assay. Results: Chromosomal analysis showed a normal metaphase spread and 46XX karyotype. The proliferation rate of endometrial CD146(+) cells in the presence of 0, 12.5, 25, 50 and 100 ng/ml SCF were 0.945±0.094, 0.962±0.151, 0.988±0.028, 1.679±0.012 and 1.129±0.145 respectively. There was a significant increase in stem/ stromal cell proliferation following in vitro treatment by 50 ng/ml than other concentrations of SCF (p=0.01). Conclusion: The present study suggests that SCF could have effect on the proliferation and cell survival of human endometrial CD146(+) cells and it has important implications for medical sciences and cell therapies.
... 5 Most of these effects were attributable to the regulation of the expression levels of transcription factors, thereby modifying a multitude of genes and modulating or achieving the phenotypic switch of VSMCs. 6 Due to the tremendous interest in this field of cellular gene regulation, new members are continuously added to this list of miRNAs (miR-663, 7 miR-34c, 8 and miR-195 9 ) holding the promise of modulating vascular remodelling during atherosclerosis and neointima formation. 10 Hu et al. 11 added miR-1298 to this ever expanding list. ...
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This editorial refers to ‘MicroRNA-1298 is regulated by DNA methylation and affects vascular smooth muscle cell function by targeting connexin 43’ by W. Hu et al. , pp. 534–545. Atherosclerosis constitutes the leading cause of cardiovascular disease resulting in ischaemia of dependent organs due to arterial occlusion. It affects not only heart and brain, but also limbs and other organs with fatal consequences for the individuum. The process invokes profound remodelling of the entire vascular wall driven by inflammation, which affects endothelial cells and promotes the activation of vascular smooth muscle cells (VSMCs). In healthy vessels, VSMCs retain a quiescent, contractile state but in response to environmental cues (chemical and mechanical) they adopt an activated, synthetic state characterized also by proliferation and migration. This remarkable plasticity is achieved through a substantial change in gene expression profile, which is likely governed by epigenetic and/or transcriptional control mechanisms. In addition to transcriptional regulation, non-coding, single-stranded, small RNAs (typically ∼22 nucleotides) contribute substantially to the phenotypic modulation of VSMCs through post-transcriptional regulation of gene expression (mRNA degradation or translational repression).1,2 These so-called microRNAs (miRNAs/miRs) promote or inhibit the switch of VSMCs to the synthetic state. For example, the miR-143/145 cluster supports a contractile state and was down-regulated by vascular injury, implicating that its lack fosters the synthetic state.3,4 Conversely, other miRNAs (miR-21, miR-221/222) were found to promote the synthetic …
... The miRNAs are critical regulators of many biological processes. The miRNAs exert their function by modulating expression of genes at the post-transcription level (Choe et al., 2015). Proliferation and differentiation are mutually exclusive during myogenesis, and miRNAs are critical for balancing these two processes (Kovanda et al., 2014). ...
Article
Pig is a useful medical model for humans due to its similarity in size and physiology. Skeletal muscle plays an essential role in body movement. However, the skeletal muscle injuries are common. Skeletal muscle function maintenance largely depends on preserving the regenerative capacity of muscle. Muscle satellite cells proliferation plays an essential role in postnatal muscle growth and regeneration. Therefore, understanding the mechanisms associated with muscle satellite cells proliferation is essential for devising the alternative treatments for muscle injury. Previous studies showed JAG1-Notch1 signaling pathway and miRNAs regulate the skeletal muscle development. JAG1-Notch1 signal pathway regulates the transcription of certain types of miRNAs which further affects target gene expression. However, the specific relationship between JAG1-Notch1 signal pathway and miRNAs during muscle development has not been established. We found overexpression of intracellular domain of the Notch1 protein (N1ICD) in porcine muscle satellite cells (PSCs) decreased miR-199b level. We demonstrated that miR-199b inhibits PSCs proliferation using the overexpression and inhibition of miR-199b experiment. We also found JAG1, the miR-199b target gene, promotes PSCs proliferation through activating the Notch1 signal pathway. Furthermore, we demonstrated miR-199b forms a feedback loop with the JAG1-Notch1 signal pathway to maintain the PSCs niche homeostasis. Our results of miRNAs and genes work collaboratively in regulating PSCs proliferation expand our understanding in PSCs proliferation mechanism. Furthermore, this finding indicates miR-199b is a potential therapeutic target for muscle atrophy.
... e results of co-expression network analysis and RNA-seq analysis showed that co-expressed miRNA networks with specific genes and pathways are related to STC [46,47]. It has been reported that exosomal miR-34c-5p reduced ICC viability by targeting the SCF/c-Kit pathway in the colon [48] and can restrain vascular smooth muscle cell multiplication and neointimal proliferation [49]. As a widely distributed miRNA, miR-34 can mediate differentiation [50], cell apoptosis [51], multiplication [52], ageing [53], and modulate various signal pathways, including transforming growth factor-β (TGF-β) pathway [51] and Wnt pathway [54]. ...
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Background: The pathogenesis of slow transit constipation (STC) is associated with exosomal miR-34c-5p. Electroacupuncture (EA) improves gastrointestinal motility in gastrointestinal disorders, especially STC. Our study aimed to explore the mechanism by which EA improves intestinal motility by modulating the release of exosomes and the transmission of exosomal miR-34c-5p. Methods: Fifty rats were randomly divided into five groups. STC model rats were induced, and GW4869, the exosome release inhibitor, was used to inhibit the release of exosome. The serum exosomes were authenticated under a transmission electron microscope and nanoparticle tracking analysis. RT-qPCR detected the expression of miR-34c-5p in serum exosomes and colonic tissues. The fecal number in 24 hours, Bristol scores, and intestinal transit rates were used to assess intestinal motility. Subsequently, hematoxylin and eosin (H&E) staining was used to examine the colonic mucosal histology. Finally, the expression of stem cell factor (SCF) and receptor tyrosine kinase (c-Kit) protein was measured using immunohistochemistry staining. Results: We found that EA upregulated exosomal miR-34c-5p in serum and downregulated miR-34c-5p in colonic tissues (P < 0.01). EA improved fecal numbers in 24 hours, Bristol scores, and intestinal transit rates in STC rats (P < 0.01). EA recovered the colonic histological structure and enhanced the expression of SCF and c-Kit protein (P < 0.01). The therapeutic effect of EA was attenuated after inhibiting the release of the exosome. Conclusion: Our results indicated that EA improves intestinal motility in STC rats by transporting of exosomal miR-34c-5p targeting the SCF/c-Kit signaling pathway.
... There is increasing evidence to suggest that miRNAs Recent studies have demonstrated that miR-34c regulates proliferation in multiple cell types, including colorectal cancer cells , glioma cells (Wu et al. 2013), hepatocellular carcinoma cells (Song et al. 2015), vascular smooth muscle cells (Choe et al. 2015), and mouse C2C12 myoblast cells (Wang et al. 2017). As mentioned previously, in breast cancer cells , and miR-590 in human umbilical cord mesenchymal stem cells (Liu et al. 2017). ...
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MicroRNAs (miRNAs) are implicated in swine spermatogenesis via their regulations of cell proliferation, apoptosis, and differentiation. Recent studies indicated that miR-34c is indispensable in the late steps of spermatogenesis. However, whether miR-34c plays similar important roles in immature porcine Sertoli cells remain unknown. In the present study, we conducted two experiments using a completely randomised design to study the function roles of miR-34c. The results from experiment I demonstrated that the relative expression level of miR-34c in swine testicular tissues increased (P=0.0017) quadratically with increasing age, while the relative expression level of SMAD family member 7 (SMAD7) decreased (P=0.0009) with curve. Furthermore, miR-34c expression levels showed a significant negative correlation (P=0.013) with SMAD7 gene expression levels. The results from experiment II indicated that miR-34c directly targets the SMAD7 gene using a luciferase reporter assay, and suppresses (P<0.05) SMAD7 mRNA and protein expressions in immature porcine Sertoli cells. Overexpression of miR-34c inhibited (P<0.05) proliferation and enhanced (P<0.05) apoptosis in the immature porcine Sertoli cells, which was supported by the results from the Cell Counting Kit-8 (CCK-8) assay, the 5-Ethynyl-2'-deoxyuridine (EdU) assay, and the Annexin V-FITC/PI staining assay. Furthermore, knockdown of SMAD7 via small interfering RNA (siRNA) gave a similar result. It is concluded that miR-34c inhibits proliferation and enhances apoptosis in immature porcine Sertoli cells by targeting the SMAD7 gene.
... It was also demonstrated that miR-125b directly targets TAZ in hepatocellular carcinoma cells, suggesting another way by which this miRNA fine-tunes the maturation of the osteoblasts [226]. In line with this consideration, it was reported that the stem cell factor (SCF) protects the osteoblasts from oxidative stress [227] and that this factor is a direct target of miR-34c (Figure 7) [228]. ...
Article
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The formation of the skeleton occurs throughout the lives of vertebrates and is achieved through the balanced activities of two kinds of specialized bone cells: the bone-forming osteoblasts and the bone-resorbing osteoclasts. Impairment in the remodeling processes dramatically hampers the proper healing of fractures and can also result in malignant bone diseases such as osteosarcoma. MicroRNAs (miRNAs) are a class of small non-coding single-strand RNAs implicated in the control of various cellular activities such as proliferation, differentiation, and apoptosis. Their post-transcriptional regulatory role confers on them inhibitory functions toward specific target mRNAs. As miRNAs are involved in the differentiation program of precursor cells, it is now well established that this class of molecules also influences bone formation by affecting osteoblastic differentiation and the fate of osteoblasts. In response to various cell signals, the tumor-suppressor protein p53 activates a huge range of genes, whose miRNAs promote genomic-integrity maintenance, cell-cycle arrest, cell senescence, and apoptosis. Here, we review the role of three p53-related miRNAs, miR-34c, -125b, and -203, in the bone-remodeling context and, in particular, in osteoblastic differentiation. The second aim of this study is to deal with the potential implication of these miRNAs in osteosarcoma development and progression.
... Vascular smooth muscle cells (VSMCs) are a crucial component of the vascular system. Under normal circumstances, VSMCs are in a homeostatic, non-proliferative state, which is mainly responsible for maintaining vascular tension and blood pressure [7,8]. In the case of damage, VSMCs interacted with a variety of cytokines released by inflammatory cells and endothelial cells [9], leading to the massive proliferation and migration of VSMCs, which is one of the multiple processes for the progression of atherosclerotic diseases. ...
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Background Atherosclerosis is the main cause of carotid artery stenosis (CAS) which mostly occurs in the elderly. In this paper, the expression level of miR-375-3p in asymptomatic CAS patients and its diagnostic value for asymptomatic CAS were investigated, and the effects of miR-375-3p on the cell proliferation and migration of vascular smooth muscle cells (VSMCs) was further explored. Methods 98 healthy subjects and 101 asymptomatic CAS patients were participated in this study. qRT-PCR was used to measure the expression level of serum miR-375-3p, and the ROC curve was established to evaluate the predictive value of miR-375-3p for asymptomatic CAS. After transfection with miR-375-3p mimic or inhibitor in vitro, cell proliferation and migration were detected by CCK-8 assay, colony formation assay, and Transwell assay, respectively. The levels of TNF-α, IL-1β, IL-6 were detected by ELISA. Western blot was used to detect the protein expression of XIAP. Finally, luciferase reporter gene assay was applied to assess the interaction of miR-375-3p with target genes. Results The expression level of serum miR-375-3p in asymptomatic CAS patients was significantly higher than that in healthy controls, and the AUC value of ROC curve was 0.888. The sensitivity and specificity were 80.2 and 86.7%, respectively, indicating that miR-375-3p had high diagnostic value for asymptomatic CAS. In vitro cell experiments showed that up-regulation of miR-375-3p significantly promoted the proliferation and migration of VSMCs, and also promoted the generation of inflammatory factors and phenotypic transformation of VSMCs. Luciferase reporter gene assay confirmed that XIAP was a target gene of miR-375-3p and was negatively regulated by miR-375-3p. Conclusions In this study, miR-375-3p may have a clinical diagnostic value for asymptomatic CAS patients which need further validation. Increased miR-375-3p levels in CAS may be associated with increased proliferation and migration of VSMCs via downregulation of the apoptosis inducing gene XIAP.
... Yi showed that miR-520 affects the proliferation, migration, and apoptosis in breast cancer cells [27]. Choe et al. suggested that miR-34 suppresses the ERK 1/2-involved pathway in VSMCs [28]. Hermeking reviewed the role of miR-34 in the apoptotic response of normal and tumor cells [29]. ...
Article
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Background In-stent restenosis usually occurs by platelet activation, neointima formation, VSMC migration, and proliferation in the position of the vessel stent. The monocytes have a magnificent role in neointimal hyperplasia since these cells recruit to the site of vessel injury through chemokines and other secretion proteins. This study is focused on the investigation of vitronectin, miR-193, miR-34, and miR-520 expression levels in PBMCs isolated from stenosed patients.MethodsA total of sixty subjects undergoing coronary artery angiography containing patients with stent no restenosis (n = 20), in-stent restenosis (n = 20), and healthy participants (n = 20) participated in the study. The vitronectin, miR-193, miR-34, and miR-520 expression levels were measured by the RT-qPCR technique. Data were analyzed by SPSS software.ResultsThe vitronectin, miR-34, and miR-520 expression levels changed significantly in patients with vessel in-stent restenosis (p = 0.02, p = 0.02, and p = 0.01, respectively). Furthermore, there were inverse correlations between the expression levels of vitronectin gene and miR-34 (r = – 0.44, p = 0.04) as well as miR-520 (r = – 0.5, p=0.01).Conclusions The molecular events in the vessel stenosis may be affected by targeting vitronectin with miR-520 and miR-34.
... Studies have shown that miR-34a is significantly increased during the differentiation of stem cells in smooth muscle cells, and it has been found that miR-34a plays an important role in promoting the differentiation of smooth muscle cells [11]. In addition, in terms of inhibiting phenotypic transformation of VSMC, early studies have found that miR-34c inhibited angiogenic intimal hyperplasia by regulating the PI3K/Akt cell signaling pathway [12]. However, the role of miR-34c in the phenotypic transformation and proliferation of PDGF-BB-induced HA-VSMC remains unknown. ...
Article
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The purpose of this study was to explore the effect of miR-34c on PDGF-BB-induced HAVSMCs phenotypic transformation and proliferation via PDGFR-β/SIRT1 pathway, so as to find a new method for early diagnosis and treatment of cardiovascular disease. HA-VSMCs were treated with platelet-derived growth factor-BB (PDGF-BB) at 0 h, 12 h, 24 h, 48 h or 36 h to explore the optimal time for phenotypic transformation of VSMCs. And then, PDGF-BB-induced HA-VSMCs were transfected with miR-34c mimics/mimics NC and pcDNA3.1-PDGFR-β/pcDNA3.1-NC to observe cell biological behaviour. CCK8 was used to detect cell proliferation activity. Transwell chamber assay was used to detect cell invasion. Early apoptosis was analyzed by flow cytometry. The expression of α-SMA and Smemb was detected by immunofluorescence staining. The expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 were analyzed by Western blot analysis. The expression of miR-34a, miR-34b and miR-34c was detected by RT-PCR, and the targeting relationship between miR-34c and PDGFR-β was detected by luciferase reporting assay. The results indicated the proliferation and migration of PDGF-BB-induced HA-VSMCs significantly increased, and apoptosis significantly decreased. Besides, α-SMA decreased significantly, while Smemb increased significantly. Furthermore, expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 increased significantly, and SIRT1 decreased significantly. Experimental results showed that, miR-34c mimics significantly inhibited cell proliferation and migration, and promoted cell apoptosis, and miR-34c inhibitor had the opposite effects. MiR-34c mimics significantly increased α-SMA expression and decreased Smemb expression, while the opposite effects were reflected after transfection with miR-34c inhibitor. Moreover, miR-34c mimics significantly decreased the expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1, and significantly increased the expression of SIRT1, while miR-34c inhibitor had the opposite effects. Luciferase assay confirmed that PDGFR-β was a potential target of miR-34c. Subsequently, PDGF-BB-induced HA-VSMCs were co-transfected with miR-34c mimics and pcDNA3.1-PDGFR-β. The results indicated that PDGFR-β reversed the biological function of miR-34c mimic. The results revealed the potential application value of miR-34c as a marker molecule of phenotypic transformation, providing a potential target for improving phenotypic transformation.
... And the single nucleotide polymorphism (SNP) in miRNA binding locations has been reported to possibly change the level of miRNA expression 13 . It was also discovered that miR-34b hindered osteoblast expansion in mice through targeting Satb2 Bae presented that miR-34b/c participated in bone tissue homeostasis, partly by regulating Notch signaling 14,15 . Moreover, a SNP named rs55763075 was found in the miR-34b binding site on MTHFR gene. ...
Article
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This study aimed to investigate the association between cognitive impairment after general anesthesia and rs55763075 polymorphisms. We enrolled and grouped patients undergoing general anesthesia according to their genotypes of rs55763075 polymorphism. Mini–Mental State Examination (MMSE) scoring was performed to evaluate the cognitive status of patients. Quantitative real-time PCR was carried out to analyze the expression of methylenetetrahydrofolate reductase (MTHFR) mRNA and miR-34b while Western blot was performed to evaluate the expression of MTHFR protein. Furthermore, we studied the effect of rs55763075 polymorphism on the expression of MEHFR via luciferase assay. Accordingly, we found that the MMSE score in GG/GA groups was significantly higher than that in AA group. And a significant reduction of MTHFR mRNA expression was observed in the serum and peripheral blood mononuclear cells (PBMCs) of patients carrying AA genotype compared with the patients carrying GG/GA genotypes. Moreover, the MTHFR expression was much lower in the cultured AA-genotyped cells transfected with miR-34b. Luciferase assay results also showed that miR-34b transfection reduced luciferase activity in the cells carrying A allele but not in cells carrying G allele. In summary, the data of this study showed that minor allele (A) of rs55763075 polymorphisms in the 3'-untranslated region of MTHFR mRNA generated a potential binding site for miR-34b, which led to reduced level of folic acid in the patients carrying the AA genotype. Furthermore, we found that the MMSE score of AA-genotyped patients was lower than that of patients carrying GG/GA genotypes.
... 7 We found that miR-132, miR-34c and miR-124 modulate VSMC proliferation and thereby affect atherogenesis. [8][9][10] Vascular calcification is another disease that is tightly regulated by miRNA. Indeed, miRNAs may function as negative regulators in vascular calcification 11,12 or as positive initiators. ...
Article
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Calcium deposition in vascular smooth muscle cells (VSMCs) is a form of ectopic ossification in blood vessels. It can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that the microRNA miR‐134‐5p potentiates inorganic phosphate (Pi)‐induced calcium deposition in VSMCs by inhibiting histone deacetylase 5 (HDAC5). Using miRNA microarray analysis of Pi‐treated rat VSMCs, we first selected miR‐134‐5p for further evaluation. Quantitative RT‐PCR confirmed that miR‐134‐5p was increased in Pi‐treated A10 cells, a rat VSMC line. Transfection of miR‐134‐5p mimic potentiated the Pi‐induced increase in calcium contents. miR‐134‐5p increased the amounts of bone runt‐related transcription factor 2 (RUNX2) protein and bone morphogenic protein 2 (BMP2) mRNA in the presence of Pi but decreased the expression of osteoprotegerin (OPG). Bioinformatic analysis showed that the HDAC5 3′untranslated region (3′UTR) was one of the targets of miR‐134‐5p . The luciferase construct containing the 3′UTR of HDAC5 was down‐regulated by miR‐134‐5p mimic in a dose‐dependent manner in VSMCs. Overexpression of HDAC5 mitigated the calcium deposition induced by miR‐134‐5p . Our results suggest that a Pi‐induced increase of miR‐134‐5p may cause vascular calcification through repression of HDAC5.
... [9][10][11][12] Our laboratory also found that miR-132, miR-34c, and miR-124 regulate atherosclerosis by targeting Lrrfip-1, stem cell factor, and S100 calcium-binding protein A4, respectively. [13][14][15] Of note, among those miRNAs, the miR-27 family has been appreciated as one of the diagnostic or prognostic markers in vascular inflammation. 16 Indeed, miR-27 is involved in angiogenesis, apoptosis, lipid regulation, and cytokine production, mainly in the endothelium, which contributes to the development of atherosclerosis. ...
Article
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Vascular calcification, the ectopic deposition of calcium in blood vessels, develops in association with various metabolic diseases and atherosclerosis and is an independent predictor of morbidity and mortality associated with these diseases. Herein, we report that reduction of microRNA-27a-3p (miR-27a-3p) causes an increase in activating transcription factor 3 (ATF3), a novel osteogenic transcription factor, in vascular smooth muscle cells. Both microRNA (miRNA) and mRNA microarrays were performed with rat vascular smooth muscle cells, and reciprocally regulated pairs of miRNA and mRNA were selected after bioinformatics analysis. Inorganic phosphate significantly reduced the expression of miR-27a-3p in A10 cells. The transcript level was also reduced in vitamin D3-administered mouse aortas. miR-27a-3p mimic reduced calcium deposition, whereas miR-27a-3p inhibitor increased it. The Atf3 mRNA level was upregulated in a cellular vascular calcification model, and miR-27a-3p reduced the Atf3 mRNA and protein levels. Transfection with Atf3 could recover the miR-27a-3p-induced reduction of calcium deposition. Our results suggest that reduction of miR-27a-3p may contribute to the development of vascular calcification by de-repression of ATF3.
... Recent studies reported that miRNA expressions, especially those of miR-132 [28], miR-34 c [29], miR-221 and miR-222 [30], were dysregulated in a rat carotid artery injury model, which attenuated neointimal hyperplasia. Previous studies revealed that various types of drugs, such as Tongxinluo [31] and Emodin [32], inhibited neointimal hyperplasia in animal model of vascular injury by regulating miRNAs expression. ...
Article
Excessive migration and proliferation of vascular smooth muscle cells (VSMCs) are critical cellular events that lead to intimal hyperplasia in atherosclerosis and restenosis. In this study, we investigated the protective effects of ursodeoxycholic acid (UDCA) on intimal hyperplasia and VSMC proliferation and migration, and the underlying mechanisms by which these events occur. A rat unilateral carotid artery was ligated to induce vascular injury and the microRNA (miRNA) expression profiles were determined using miRNA microarray analysis. We observed that UDCA significantly reduced the degree of intimal hyperplasia and induced miR-21 dysregulation. Restoration of miR-21 by agomir-miR-21 reversed the protective effects of UDCA on intimal hyperplasia and proliferation in vivo. In vitro, UDCA suppressed PDGF-BB-induced VSMC proliferation, invasion and migration in a dose-dependent manner, whereas the suppressive effect of UDCA was abrogated by overexpression of miR-21 in PDGF-BB-incubated VSMCs. Furthermore, we identified that miR-21 in VSMCs targeted the phosphatase and tensin homolog (PTEN), a tumor suppressor gene, negatively modulated the AKT/mTOR pathway. More importantly, we observed that that UDCA suppressed AKT/mTOR signaling pathway in the carotid artery injury model, whereas this pathway was reactivated by overexpression of miR-21. Taken together, our findings indicated that UDCA inhibited intimal hyperplasia and VSMCs excessive migration and proliferation via blocking miR-21/PTEN/AKT/mTOR signaling pathway, which suggests that UDCA may be a promising candidate for the therapy of atherosclerosis.
... miR-34a can also promote the proliferation of human pulmonary artery smooth muscle cells [15]. In addition, miR-34a and miR-34c could inhibit the proliferation of smooth muscle cells [16,17]. miR-34c was also reported to repress the proliferation of muscle satellite cells while promoting the differentiation process [18]. ...
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As key post-transcriptional regulators, microRNAs (miRNAs) play an indispensable role in skeletal muscle development. Our previous study suggested that miR-34b-5p and IGFBP2 could have a potential role in skeletal muscle growth. Our goal in this study is to explore the function and regulatory mechanism of miR-34b-5p and IGFBP2 in myogenesis. In this study, the dual-luciferase reporter assay and Western blot analysis showed that IGFBP2 is a direct target of miR-34b-5p. Flow cytometric analysis and EdU assay showed that miR-34b-5p could repress the cell cycle progression of myoblasts, and miR-34b-5p could promote the formation of myotubes by promoting the expression of MyHC. On the contrary, the overexpression of IGFBP2 significantly facilitated the proliferation of myoblasts and hampered the formation of myotubes. Together, our results indicate that miR-34b-5p could mediate the proliferation and differentiation of myoblasts by targeting IGFBP2.
... МикроРНК-34c был идентифицирован как новый модулятор, который ингибирует деление ГМК и неоинтиму, действуя на SCF. МикроРНК-34c уменьшает фосфорилирование ERK и увеличивает экспрессию KLF4, p21, p27 и Bax, что предполагает участие в регуляции ГМК сигнального пути SCF/ERK [9]. ...
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Цукровий діабет і атеросклероз: епігенетичні механізми патогенезу. Огляд літератури Л.К. Соколова, В.М. Пушкарьов, О.I. Ковзун, В.В. Пушкарьов, М.Д. Тронько В огляді літератури узагальнено і проаналізовано матеріал, присвячений епігенетичним змінам, які супроводжують серцево-судинні ускладнення при цукровому діабеті. Представлено дані про участь епігенетичних модифікацій у патологічних змінах клітин ендотелію, гладеньком’язової мускулатури і макрофагів, що ведуть до атеросклерозу. Описана роль різних мікроРНК у диференціюванні, активації, запаленні, проліферації і міграції клітин судин. Показано, що модифікації гістонів, метилювання ДНК і зміна спектра мікроРНК беруть участь в ініціації і розвитку серцево-судинних захворювань при цукровому діабеті, а їх вивчення і застосування отриманих знань має великий діагностичний, прогностичний, а в перспективі і терапевтичний потенціал. Ключові слова: атеросклероз, цукровий діабет, епігенетичні модифікації, мікроРНК.
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Background and purpose: The anti-helminthic drug niclosamide regulates multiple cellular signals including STAT3, AMPK, Akt, Wnt/β-catenin, and mitochondrial uncoupling which involved in neointimal hyperplasia. Therefore, the aim of the present study is to examine the effects of the niclosamide on vascular smooth muscle cell proliferation, migration, and neointimal hyperplasia and elucidate the potential mechanisms. Experimental approach: Cell migration was measured by using wound-induced migration assay and Boyden chamber assay. Protein levels were measured by using western blot technique. Neointimal hyperplasia in vivo was obtained by subjecting rats to carotid artery balloon injury. Key results: Niclosamide treatment inhibited serum (15% FBS)- and platelet-derived growth factor-BB (PDGF-BB)-induced proliferation and migration of vascular smooth muscle cells (A10 cells). Niclosamide showed no apparent cytotoxicity at anti-proliferative concentrations, but induced cell apoptosis when the concentration was increased. Niclosamide treatment inhibited serum (15% FBS)- and PDGF-BB-induced STAT3 activation (increased protein levels of p-STAT3 at Tyr705), activated AMPK in A10 cells. Niclosamide showed no significant effect on β-catenin expression and the activities of ERK1/2 and Akt in A10 cells. Intraperitoneal injection of soluble pegylated niclosamide (PEG5000-niclosamide) (equivalent to niclosamide 25 mg/kg) attenuated neointimal hyperplasia in balloon-injured rat carotid arteries in vivo. Conclusions and implications: Niclosamide inhibits vascular smooth muscle cell proliferation and migration, and attenuates neointimal hyperplasia in balloon-injured rat carotid arteries through a mechanism involving inhibition of STAT3.
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High glucose-insulted bone marrow-derived mesenchymal stem cells (BMCs) showed impaired angiogenesis along with downregulation of stem cell factor (SCF). This study was designed to determine the involvement of microRNAs (miR), which are actively involved in the physiological function of stem cells. We observed that miR-34c was significantly induced by high glucose treatment and blunted tube formation of BMCs. Stem cell factor (SCF) was confirmed as a target of miR-34c by 3'-UTR promoter analysis and Western blot. SCF knockdown by siRNA induced Krüppel-like factor 4 (KLF4) and resulted in the blockade of angiogenesis of BMCs. Sequentially, KLF4 overexpression completely blocked tube formation through inducing PAI-1 (plasminogen activator inhibitor-1). To study the action of miR-34c in terms of the therapeutic potential of BMCs, myocardial infarction (MI) was induced by ligation of the coronary artery in nude mice, BMCs transfected with miR-control of miR-34c were injected into the infarcted myocardium 7 days later, and histological studies were performed 2 weeks later. Cardiac fibrosis was 18.24±4.7% in the miR-34c-BMC group and 10.01±0.2% in the miR-control-BMC group (p<0.05). Cardiac function and vessel density were decreased in the miR-34c-BMC group compared with the miR-con-BMC group. Particularly, miR-34c-BMCs failed to incorporate into vessels. Our results show that the angiogenic activity of BMCs is finely regulated by the miR-34c-SCF-KLF4 axis, which is a potent translational target for optimizing the therapeutic activity of autologous BMCs for cardiac repair. Copyright © 2015. Published by Elsevier Inc.
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Arterial vascular disease causes carotid stenosis, peripheral vascular disease, and coronary artery disease and is the leading cause of death in the western world. There are various manifestations, histomorphological structures, etiologies, risk factors and complications arising from this disease complex. Although different diseases with distinct features have been defined, they all have in common that they are manifested in the arterial vascular system and can be summarized under the umbrella term of arterial vascular disease. This article summarizes the current knowledge on arterial vascular disease and discusses various novel preventive and therapeutic strategies that target smooth muscle cell proliferation.
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Vascular smooth muscle cells (VSMCs) hyperplasia is a common feature of pathologic cardiovascular event such as restenosis and atherosclerosis. The role and mechanisms of microRNAs (miRs) in VSMCs proliferation are poorly understood. Here, we report that miR-181b promotes VSMCs proliferation and migration. In an animal model, miR-181b was significantly increased in the rat carotid artery after balloon catheter injury. Delivery of miR-181b inhibitor to injured artery exhibited a marked inhibition of neointimal hyperplasia. Transfection of miR-181b with "mimics" to A10 cells accelerated cell proliferation, which was accompanied by an increase of cell migration. The induction of A10 cells proliferation by miR-181b appeared to be involved in activation of S and G2/M checkpoint, concomitant with decreases in cell-cycle inhibitors p21 and p27, and increases in cell-cycle activators CDK4 and cyclinD1. In contract, miR-181b inhibition attenuated A10 cells proliferation, inhibited cell migration and arrested cell cycle transition. Moreover, forced miR-181b expression elevated the phosphorylation levels of Akt and Erk1/2, whereas inhibition of miR-181b produced the opposite effects. Additionally, inhibition of PI3K and MAPK signaling pathways with specific inhibitors, but not inhibition of JNK pathway, significantly abolished the effects of miR-181b in promoting cell proliferation. These findings demonstrate that miR-181b enhances the proliferation and migration of VSMCs through activation of PI3K and MAPK pathways.
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MicroRNAs are a group of endogenously small non-coding RNA molecules that downregulate gene expression at the post-transcriptional level through binding to the 3’UTR of target mRNAs. Recent findings have revealed a key role for microRNAs in the pathophysiological processes of atherosclerosis. As a complex disease, atherosclerosis is influenced by a combination of multiple genes and environmental factors. Both of them play a role in atherogenesis by affecting different types of cells (such as endothelial cell, vascular smooth muscle cell and monocyte/macrophage) function. MicroRNAs control the senescence and dysfunction of endothelial cells, proliferation and migration of vascular smooth muscle cells, and macrophage-driven cytokine production and polarization. By these effects, microRNAs can influence the processes of atherosclerosis and may represent new molecular targets for therapy.
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Objective: Vascular smooth muscle cells (VSMCs) modulate their phenotype between synthetic and contractile states in response to environmental changes; this modulation plays a crucial role in the pathogenesis of restenosis and atherosclerosis. Here, we identified fibroblast growth factor 12 (FGF12) as a novel key regulator of the VSMC phenotype switch. Approach and results: Using murine models and human specimens, we found that FGF12 was highly expressed in contractile VSMCs of normal vessel walls but was downregulated in synthetic VSMCs from injured and atherosclerotic vessels. In human VSMCs, FGF12 expression was inhibited at the transcriptional level by platelet-derived growth factor-BB. Gain- and loss-of-function experiments showed that FGF12 was both necessary and sufficient for inducing and maintaining the quiescent and contractile phenotypes of VSMCs. FGF12 inhibited cell proliferation through the p53 pathway and upregulated the key factors involved in VSMC lineage differentiation, such as myocardin and serum response factor. Such FGF12-induced phenotypic change was mediated by the p38 MAPK pathway. Moreover, FGF12 promoted the differentiation of mouse embryonic stem cells and the transdifferentiation of human dermal fibroblasts into SMC-like cells. Furthermore, adenoviral infection of FGF12 substantially decreased neointima hyperplasia in a rat carotid artery injury model. Conclusions: In general, FGF family members induce a synthetic VSMC phenotype. Interestingly, the present study showed the unanticipated finding that FGF12 belonging to FGF family, strongly induced the quiescent and contractile VSMC phenotypes and directly promoted VSMC lineage differentiation. These novel findings suggested that FGF12 could be a new therapeutic target for treating restenosis and atherosclerosis.
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Background Vascular smooth muscle cells (VSMCs) play an important role in the biosynthetic, proliferative, and contractile functions of vessel walls. Hyper-proliferation of VSMCs is a crucial event associated with the pathogenesis of various vascular diseases including hypertension, atherosclerosis, and re-stenosis. Area covered A variety of studies have been performed to identify the signaling molecular targets or inhibitory drugs to suppress hyper-proliferation of VSMCs. Emerging studies have highlighted the relevance of targeting various signaling pathways, miRNAs, and post-translational modifications in controlling the hyper-proliferation of VSMCs. Expert opinion This review will introduce various inhibitors, a wide range of miRNAs, and different histone deacetylases related to the molecular targets and underlying signaling pathways involved in the regulation of VSMC hyper-proliferation. This will contribute to the development of promising approaches for the treatment of vascular diseases.
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Vascular smooth muscle cell (VSMC) proliferation and migration are vital to atherosclerosis (AS) development and plaque rupture. MicroRNA-377-3p (miR-377-3p) has been reported to inhibit AS in apolipoprotein E knockout (ApoE-/-) mice. Herein, the mechanism underlying the effect of miR-377-3p on alleviating AS is explored. In vivo experiments, ApoE-/- mice were fed with high-fat diet (HFD) to induce AS and treated with miR-377-3p agomir or negative control agomir (agomir-NC) on week 0, 2, 4, 6, 8, 10 after HFD feeding. MiR-377-3p was found to restore HFD-induced AS lesions and expressions of matrix metalloproteinase (MMP)-2, MMP-9, α-smooth muscle actin (α-actin) and calponin. In vitro experiments, human VSMCs were tranfected with miR-377-3p agomir or agomir-NC, followed by treatment with oxidized low-density lipoprotein (ox-LDL). MiR-377-3p was observed to significantly inhibit ox-LDL-induced VSMC proliferation characterized by inhibited cell viability, expressions of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E and cell cycle transition from G1 to S phase accompanied with less 5-Ethynyl-2’-deoxyuridine (EdU)-positive cells. Furthermore, MiR-377-3p significantly inhibited ox-LDL-induced VSMC migration characterized by inhibited wound closure and decreased relative VSMC migration. Besides, neuropilin2 (NRP2) was verified as a target of miR-377-3p. MiR-377-3p was observed to inhibit NRP2 expressions in vivo and in vitro. Moreover, miR-377-3p significantly inhibited MMP-2 and MMP-9 expressions in human VSMCs. Additionally, miR-377-3p-induced inhibition of VSMC proliferation and migration could be attenuated by NRP2 overexpression. These results indicated that miR-377-3p inhibited VSMC proliferation and migration via targeting NRP2. This study provides an underlying mechanism for miR-377-3p-based AS therapy.
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Purpose: Microvesicles (MV) can modulate the function of recipient cells by transferring their contents. Our previous study highlighted that MV released from tumor necrosis factor-α (TNF-α) plus serum deprivation (SD)-stimulated endothelial progenitor cells, induce detrimental effects on endothelial cells. In this study, we investigated the potential effects of endothelial MV (EMV) on proliferation, migration, and apoptosis of human brain vascular smooth cells (HBVSMC). Methods: EMV were prepared from human brain microvascular endothelial cells (HBMEC) cultured in a TNF-α plus SD medium. RNase-EMV were made by treating EMV with RNase A for RNA depletion. The proliferation, apoptosis and migration abilities of HBVSMC were determined after co-culture with EMV or RNase-EMV. The Mek1/2 inhibitor, PD0325901, was used for pathway analysis. Western blot was used for analyzing the proteins of Mek1/2, Erk1/2, phosphorylation Erk1/2, activated caspase-3 and Bcl-2. The level of miR-146a-5p was measured by qRT-PCR. Results: (1) EMV significantly promoted the proliferation and migration of HBVSMC. The effects were accompanied by an increase in Mek1/2 and p-Erk1/2, which could be abolished by PD0325901; (2) EMV decreased the apoptotic rate of HBVSMC by approximately 35%, which was accompanied by cleaved caspase-3 down-regulation and Bcl-2 up-regulation; (3) EMV increased miR-146a-5p level in HBVSMC by about 2-folds; (4) RNase-treated EMV were less effective than EMV on HBVSMC activities and miR-146a-5p expression. Conclusion: EMV generated under inflammation challenge can modulate HBVSMC function and fate via their carried RNA. This is associated with activation of theMek1/2/Erk1/2 pathway and caspase-3/Bcl-2 regulation, during which miR-146a-5p may play an important role. The data suggest that EMV derived from inflammation-challenged endothelial cells are detrimental to HBVSMC homeostatic functions, highlighting potential novel therapeutic targets for vascular diseases.
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Ischemic stroke resulting from atherosclerosis (particularly in the carotid artery) is one of the major subtypes of stroke and has a high incidence of death. Disordered lipid homeostasis, lipid deposition, local macrophage infiltration, smooth muscle cell proliferation, and plaque rupture are the main pathological processes of atherosclerotic ischemic stroke. Hepatocytes, macrophages, endothelial cells and vascular smooth muscle cells are the main cell types participating in these processes. By inhibiting the expression of the target genes in these cells, microRNAs play a key role in regulating lipid disorders and atherosclerotic ischemic stroke. In this article, we listed the microRNAs implicated in the pathology of atherosclerotic ischemic stroke and aimed to explain their pro- or antiatherosclerotic roles. Our article provides an update on the potential diagnostic use of miRNAs for detecting growing plaques and impending clinical events. Finally, we provide a perspective on the therapeutic use of local microRNA delivery and discuss the challenges for this potential therapy.
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Vascular smooth muscle cell (VSMC) proliferation and migration are crucial events in the pathological course of atherosclerosis and restenosis after percutaneous coronary intervention (PCI). Dioscin has been shown to exhibit powerful cardiovascular protective effects and potent therapeutic potential in cancer owing to the inhibition of cell proliferation and migration. However, its effects on arterial wall hypertrophy-related diseases caused by VSMC proliferation and migration remain unclear. In this study, we investigated the effects of dioscin on intimal hyperplasia after balloon injury in vivo, its effects on VSMC proliferation and migration in vitro, and the mechanisms underlying these effects. Results showed that dioscin treatment significantly inhibited VSMC proliferation and intimal thickening after balloon injury. In cultured VSMCs, treatment with dioscin significantly decreased fetal bovine serum or platelet-derived growth factor-induced cell proliferation and migration. Moreover, dioscin inhibited the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and expression of Forkhead box M1 (FoxM1) and its downstream target genes. FoxM1 knockdown with shRNA partially counteracted the inhibitory effects of dioscin on cell proliferation and migration. In conclusion, we demonstrated that dioscin attenuated neointima formation in response to balloon injury by suppressing VSMC proliferation and migration through MAPK-FoxM1 pathway. Our data suggested that dioscin might be a potential therapeutic agent for atherosclerosis and restenosis after PCI.
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Vascular calcification refers to the pathological deposition of calcium and phosphate minerals into the vasculature. It is prevalent in atherosclerosis, ageing, type 2 diabetes mellitus and chronic kidney disease, thus, increasing morbidity and mortality from these conditions. Vascular calcification shares similar mechanisms with bone mineralization, with smooth muscle cells playing a critical role in both processes. In the last decade, a variety of microRNAs have been identified as key regulators for the differentiation, phenotypic switch, proliferation, apoptosis, cytokine production and matrix deposition in vascular smooth muscle cells during vascular calcification. Therefore, this review mainly discusses the roles of microRNAs in the pathophysiological mechanisms of vascular calcification in smooth muscle cells and describes several interventions against vascular calcification by regulating microRNAs. As the exact mechanisms of calcification remain not fully elucidated, having a better understanding of microRNA involvement in vascular calcification may give impetus to development of novel therapeutics for the control and treatment of vascular calcification.
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The inflammatory events related to prostaglandins may play an important role in the progression of vessel stenosis. The aim of this study was to investigate the monocyte PTGES and 15-PGDH gene expression levels and the serum 13,14-dihyro-15-keto-PGF2α value involved in PGE2 metabolism in patients with coronary artery stenosis and restenosis. Moreover, the effects of miR-520, miR-1297 and miR-34 were studied on the gene expression levels. A total of sixty subjects referred for coronary angiography including healthy controls (stenosis < 5%), subjects with stent no restenosis) SNR, stenosis < 5%) and subjects in stent restenosis (ISR, restenosis >70%) were participated in the study. The gene expression levels and the serum 13,14-dihyro-15-keto- PGF2α value were measured by RT-qPCR and ELISA techniques, respectively. Moreover, the effects of miRNAs on the gene expression levels were investigated by the monocyte transfection of miR/PEI complexes. The PTGES and 15-PGDH gene expression levels and serum 13,14-dihyro-15-keto- PGF2α value increased significantly (P<0.05). Based on the miR-520 and miR-34 expression levels, the miR/PEI transfection studies were confirmed significantly the gene expression changes. The monocyte PGE2 synthesis pathway is actively considered in the SNR and ISR patients and might be related to miR-34 and miR-520 functions.
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Trichosanthin (TCS), isolated from the root tuber of Trichosantheskirilowii, a well-known traditional Chinese medicinal plant, belonging to the Cucurbitaceae family, was found to exhibit numerous biological and pharmacological activities including anti-inflammatory. However, the effects of TCS on arterial injury induced neointimal hyperplasia and inflammatory cell infiltration remains poorly understood. The aim of study was to examine the effectiveness of TCS on arterial injury-mediated inflammatory processes and underlying mechanisms. A balloon-injured carotid artery induced injury in vivo in rats was established as a model of vascular injury. After 1 day TCS at 20, 40, or 80 mg/kg/day was administered intraperitoneally, daily for 14 days. Subsequently, the carotid artery was excised and taken for immunohistochemical staining. Data showed that TCS significantly dose-dependently reduced balloon injury-induced neointima formation in the carotid artery model rat, accompanied by markedly decreased positive expression percentage proliferating cell nuclear antigen (PCNA). In the in vitro study vascular smooth muscle cells (VSMC) were cultured, proliferation stimulated with platelet-derived growth factor-BB (PDGF-BB) (20 ng/ml) and TCS at 1, 2, or 4 μM added. Data demonstrated that TCS inhibited proliferation and cell cycle progression of VSMC induced by PDGF-BB. Further, TCS significantly lowered mRNA expression of cyclinD1, cyclinE1, and c-fos, and protein expression levels of Akt1, Akt2, and mitogen-activated protein kinase MAPK (ERK1) signaling pathway mediated by PDGF-BB. These findings indicate that TCS inhibits vascular neointimal hyperplasia induced by vascular injury in rats by suppression of VSMC proliferation and migration, which may involve inhibition of Akt/MAPK/ERK signal pathway.
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The transplantation of cardiac stem cells (CSCs) is thought to be responsible for improving the performance of injured heart induced by myocardial infarction (MI). However, the mechanisms involved in the migration of activated CSCs post‑MI remain to be clarified. In this study, CSCs were isolated from rat hearts and a cellular migration assay was performed using a 24‑well Transwell system. Stem cell factor (SCF) induced CSC migration in a concentration‑dependent manner, which could be blocked with an SCF antibody as well as a PI3K/AKT inhibitor, LY294002. Moreover, SCF induced the expression and activity of matrix metalloproteinase (MMP)‑2 and MMP‑9 in a concentration‑ and time‑dependent manner, as measured by quantitative RT‑PCR, western blot analysis and gelatin zymography. Results of western blot analysis revealed phosphorylated AKT was markedly increased in SCF‑treated CSCs and that inhibition of SCF/c‑Kit signaling or phospho‑AKT activity significantly attenuated the SCF‑induced expression of MMP‑2 and MMP‑9. Thus, our results showed that SCF partially mediated CSC migration via the activation of PI3K/AKT/MMP‑2/‑9 signaling.
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The proliferation and remodeling of vascular smooth muscle cells (VSMCs) is an important pathological event in atherosclerosis and restenosis. Here we report that microRNA-132 (miR-132) blocks vascular smooth muscle cells (VSMC) proliferation by inhibiting the expression of LRRFIP1 [leucine-rich repeat (in Flightless 1) interacting protein-1]. MicroRNA microarray revealed that miR-132 was upregulated in the rat carotid artery after catheter injury, which was further confirmed by quantitative real-time RT-PCR. Transfection of a miR-132 mimic significantly inhibited the proliferation of VSMCs, whereas transfection of a miR-132 antagomir increased it. miR-132 mimic inhibited VSMC migration and induced apoptosis. miR-132 mimic increased the protein amounts of both p27 and smooth muscle (SM) α-actin, whereas it decreased SM α-actin and Bcl2. Bioinformatics showed that LRRFIP1 is a target candidate of miR-132. miR-132 down-regulated luciferase activity driven by a vector containing the 3'-untranslated region of Lrrfip1 in a sequence-specific manner. LRRFIP1 induced VSMC proliferation and increased phosphorylation of ERK. Immunohistochemical analysis revealed that Lrrfip1 was clearly expressed along with the basal laminar area of smooth muscle, and its expression pattern was disrupted 7 days after arterial injury. LRRFIP1 mRNA was decreased 14 days after injury. Delivery of miR-132 to rat carotid artery reduced LRRFIP1 expression and attenuated neointimal proliferation in carotid artery injury models. Our results suggest that miR-132 is a novel regulator of VSMC proliferation that represses neointimal formation by inhibiting LRRFIP1 expression.
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AimsThe long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation.Methods and resultsWe undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Furthermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a significant reduction in neointimal formation.Conclusion This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft remodelling.
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Stem cell factor (SCF) is a dimeric molecule that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Activation of c-Kit leads to its autophosphorylation and initiation of signal transduction. Signaling proteins are recruited to activated c-Kit by certain interaction domains (e.g., SH2 and PTB) that specifically bind to phosphorylated tyrosine residues in the intracellular region of c-Kit. Activation of c-Kit signaling has been found to mediate cell survival, migration, and proliferation depending on the cell type. Signaling from c-Kit is crucial for normal hematopoiesis, pigmentation, fertility, gut movement, and some aspects of the nervous system. Deregulated c-Kit kinase activity has been found in a number of pathological conditions, including cancer and allergy. The observation that gain-of-function mutations in c-Kit can promote tumor formation and progression has stimulated the development of therapeutics agents targeting this receptor, e.g., the clinically used inhibitor imatinib mesylate. Also other clinically used multiselective kinase inhibitors, for instance, sorafenib and sunitinib, have c-Kit included in their range of targets. Furthermore, loss-of-function mutations in c-Kit have been observed and shown to give rise to a condition called piebaldism. This review provides a summary of our current knowledge regarding structural and functional aspects of c-Kit signaling both under normal and pathological conditions, as well as advances in the development of low-molecular-weight molecules inhibiting c-Kit function.
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MicroRNAs (miRNAs) are endogenously expressed, small noncoding RNAs that inhibit gene expression by binding to target mRNAs. Recent studies have revealed that miRNAs function as tumor suppressors or oncogenes. In the present study, we investigated the role of miRNA-34b/c in uveal melanoma. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed to detect the expression level of miR-34b/c in uveal melanoma cells and primary samples. Subsequently, uveal melanoma cell proliferation was examined by the MTS (3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl] -2H-tetrazolium, inner salt) assay, clone formation assay, and flow cytometry. Cell apoptosis was measured by caspase3/7 assay. Cell migration was evaluated by transwell migration assay. The target of miR-34b/c was predicted by bioinformatics and validated by luciferase assay. In addition, the effect of miR-34b/c on c-Met, cell cycle-related proteins, v-akt murine thymoma viral oncogene homolog (Akt) and extracellular signal-regulated kinase (ERK) pathway was determined by western blotting. miR-34b/c expression, which was dramatically decreased in uveal melanoma cells and clinical samples, can be upregulated by doxorubicin and epigenetic drugs. The transfection of miR-34b/c into uveal melanoma cells leads to a significant reduction in cell growth and migration. miR-34b/c caused cell cycle G(1) arrest rather than the induction of apoptosis. Met proto-oncogene (c-Met) was identified as a target of miR-34b/c in uveal melanoma cells. Furthermore, miR-34b/c was confirmed to downregulate the expression of c-Met, p-Akt, and cell cycle-related proteins by western blotting. Our results demonstrate that both miR-34b and miR-34c act as tumor suppressors in uveal melanoma cell proliferation and migration through the downregulation of multiple targets.
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Rationale: Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. MicroRNAs (miRNAs) have emerged as important regulators for VSMC function, and we recently identified miR-663 as critical for controlling human aortic smooth muscle cell proliferation. Objective: To investigate whether miR-663 plays a role in human VSMC phenotypic switch and the development of neointima formation. Methods and results: By using quantitative reverse-transcription polymerase chain reaction, we found that miR-663 was significantly downregulated in human aortic VSMCs on platelet-derived growth factor treatment, whereas expression was markedly increased during VSMC differentiation. Furthermore, we demonstrated that overexpression of miR-663 increased expression of VSMC differentiation marker genes, such as smooth muscle 22α, smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain, and potently inhibited platelet-derived growth factor-induced VSMC proliferation and migration. We identified the transcription factor JunB and myosin light chain 9 as downstream targets of miR-663 in human VSMCs, because overexpression of miR-663 markedly inhibited expression of JunB and its downstream molecules, such as myosin light chain 9 and matrix metalloproteinase 9. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by ≈50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression. Conclusions: These results indicate that miR-663 is a novel modulator of human VSMC phenotypic switch by targeting JunB/myosin light chain 9 expression. These findings suggest that targeting miR-663 or its specific downstream targets in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.
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microRNAs (miRNAs) play vital roles in several biological processes, including apoptosis, by negatively regulating the expression of target genes. The molecular mechanisms of the key survival signal, Akt family, have been widely explored. However, it remains to be ascertained whether Akt1, the predominant isoform in most tissue, is a direct target of miRNA. In this study, we identified Akt1 and E2F1 to be two direct targets of miR-149* and b-Myb to be an indirect target by reporter assays and Western blot analyses. Ectopic expression of miR-149*-induced apoptosis in Be2C, a neuroblastoma cell line, and in HeLa cells. Silencing of Akt1 or E2F1 expression also led to similar apoptotic changes in these two cell lines, suggesting that the pro-apoptotic effects of miR-149* were exerted by repressing Akt1 and E2F1 expressions. Importantly, analysis of primary neuroblastoma samples revealed a significant inverse correlation of miR-149* with E2F1 expressions (P=0.026). Interestingly, using the reporter assays, excess miR-149 introduced by transfection to simulated its preponderance in the in vivo condition, could not overcome the repressive function of miR-149* on the target genes. This implies that the pro-apoptotic function of miR-149* may not be dampened by its predominant cognate, miR-149, in vivo. Our findings not only provided the first evidence that Akt1 is a direct target of miRNA but also demonstrated that miR-149* is a pro-apoptotic miRNA by repressing the expression of Akt1 and E2F1.
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Aloe-emodin (AE), extracted from the rhizome of Rheum palmatum, has an anti-proliferative effect on different human cancer cell lines. Nonetheless, the underlying mechanism by which AE inhibits nasopharyngeal carcinoma (NPC) cell invasion is still unclear. The results of this study show that treatment of NPC cells with growth suppressive concentrations of AE caused cell cycle arrest at the S-G(2)/M phase. Coimmunoprecipitation and small interfering RNA (siRNA) studies demonstrated that AE-induced cell cycle arrest in NPC cells was associated with increasing levels of cyclin B1 bound to cyclin-dependent kinase 1. The inhibition of NPC cell invasion by AE was evidenced through the suppression of matrix metalloproteinases-2 (MMP-2) expression. MMP-2 promoter activity and cell invasion were inhibited by p38 mitogen-activated protein kinase (MAPK) siRNA, inhibitor 4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1H-imidazole (SB203580), and AE, but not by JNK siRNA and inhibitor 1,9-pyrazoloanthrone. Treatment with AE, SB203580, NF-kappaB inhibitors N-p-tosyl-(L)-phenylalanine chloromethyl ketone (TPCK) and pyrrolidine dithiocarbamate (PDTC) or transfection with p38 MAPK siRNA significantly inhibited NF-kappaB transcriptional activity. In addition, TPCK and PDTC treatment inhibited the expression and promoter activity of MMP-2 and thereby significantly inhibited cell invasion activity. The involvement of p38 MAPK activity in NF-kappaB-mediated MMP-2 function was further confirmed through the attenuation of p38 MAPK by SB203580 and NF-kappaB ectopic expression. Collectively, our results indicate that AE inhibits invasion of NPC cells by suppressing the expression of MMP-2 via the p38 MAPK-NF-kappaB signaling pathway.
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K562 cells and peripheral blood mononuclear cells were treated with hydrogen peroxide (H(2)O(2)) to determine the expression of Krüppel-like factor (KLF) 4. A full-length complementary DNA or an anti-sense oligonucleotide of KLF4 was transfected into cells, and expressions of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2-associated X (bax) proteins were analyzed. The results showed that H(2)O(2) treatment of cells resulted in an increase in KLF4 levels; KLF4 induced apoptosis and slowed cell growth, potentially resulting from up-regulation of bax and down-regulation of bcl-2. Transcriptional activities on bcl-2 and bax were promoted following KLF4 overexpression potentially through KLF4 binding sites on corresponding promoters. All results indicate that KLF4 induces apoptosis in leukemia cells involving the bcl-2/bax pathway during H(2)O(2) stimulation, suggesting a potential mechanism for research on drug-induced apoptosis.
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A better understanding of the molecular mechanisms that govern human adipose tissue-derived mesenchymal stem cells (hASCs) differentiation could improve hASCs-based cell therapy and provide new insights into a number of diseases, including obesity. In this study, we examined the roles of microRNA-21 (miR-21) in adipogenic differentiation of hASCs. We found that miR-21 expression was transiently increased after induction of adipogenic differentiation, peaked at 3 days, and returned to the baseline level 8 days. Lentiviral overexpression of miR-21 enhanced adipogenic differentiation. Overexpression of miR-21 decreased both protein and mRNA levels of TGFBR2. The expression of TGFBR2 was decreased during adipogenic differentiation of hASCs in concordance with an increase in the level of miR-21. In contrast, inhibiting miR-21 with 2′-O-methyl-antisense microRNA increased TGFBR2 protein levels in hASCs, accompanied by decreased adipogenic differentiation. The activity of a luciferase construct containing the miR-21 target site from the TGFBR2 3′UTR was lower in LV-miR21-infected hASCs than in LV-miLacZ infected cells. TGF-β-induced inhibition of adipogenic differentiation was significantly decreased in miR-21 overexpressing cells compared with control lentivirus-transduced cells. RNA interference-mediated downregulation of SMAD3, but not of SMAD2, increased adipogenic differentiation. Overexpression and inhibition of miR-21 altered SMAD3 phosphorylation without affecting total levels of SMAD3 protein. Our data are the first to demonstrate that the role of miR-21 in the adipogenic differentiation of hASCs is mediated through the modulation of TGF-β signaling. This study improves our knowledge of the molecular mechanisms governing hASCs differentiation, which may underlie the development of obesity or other metabolic diseases. Disclosure of potential conflicts of interest is found at the end of this article.
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Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p < or = 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p < or = 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.