International Journal of Biological Sciences

Published by Ivyspring International Publisher
Print ISSN: 1449-2288
Publications
The serine-threonine kinase CK2 exhibits genomic alterations and aberrant overexpression in human head and neck squamous cell carcinomas (HNSCC). Here, we investigated the effects of CK2 inhibitor CX-4945 in human HNSCC cell lines and xenograft models. The IC50's of CX-4945 for 9 UM-SCC cell lines measured by MTT assay ranged from 3.4-11.9 μM. CX-4945 induced cell cycle arrest and cell death measured by DNA flow cytometry, and inhibited prosurvival mediators phospho-AKT and p-S6 in UM-SCC1 and UM-SCC46 cells. CX-4945 decreased NF-κB and Bcl-XL reporter gene activities in both cell lines, but upregulated proapoptotic TP53 and p21 reporter activities, and induced phospho-ERK, AP-1, and IL-8 activity in UM-SCC1 cells. CX-4945 exhibited modest anti-tumor activity in UM-SCC1 xenografts. Tumor immunostaining revealed significant inhibition of PI3K-Akt-mTOR pathway and increased apoptosis marker TUNEL, but also induced p-ERK, c-JUN, JUNB, FOSL1 and proliferation (Ki67) markers, as a possible resistance mechanism. To overcome the drug resistance, we tested MEK inhibitor PD-0325901 (PD-901), which inhibited ERK-AP-1 activation alone and in combination with CX-4945. PD-901 alone displayed significant anti-tumor effects in vivo, and the combination of PD-901 and CX-4945 slightly enhanced anti-tumor activity when compared with PD-901 alone. Immunostaining of tumor specimens after treatment revealed inhibition of p-AKT S129 and p-AKT T308 by CX-4945, and inhibition of p-ERK T202/204 and AP-1 family member FOSL-1 by PD-901. Our study reveals a drug resistance mechanism mediated by the MEK-ERK-AP-1 pathway in HNSCC. MEK inhibitor PD-0325901 is active in HNSCC resistant to CX-4945, meriting further clinical investigation.
 
1,25-(OH)2D3 activates MMP13 expression in chondrocytes in a dose-dependent manner. Two different chondrocytes were used in these experiments: ATDC5 chondrocyte (A) and RCS chondrocyte (B). Chondrocytes were cultured in DMEM and treated with different amounts of 1,25-(OH)2D3 as indicated for 24 hr. RNA was isolated and measured by quantitative real-time RT-PCR. Level of RNA from control group was normalized to a value of 1. Values are presented as the mean ±S.D. *: A star indicates statistical significance compared to control group.
1,25-(OH)2D3 activates MMP13 expression in a time-dependent manner. ATDC5 chondrocytes were cultured in DMEM and treated with 10 nM of 1,25-(OH)2D3 for different time points as indicated. RNA was isolated and measured by quantitative real-time RT-PCR. Level of RNA from control group was normalized to a value of 1. Values are presented as the mean ±S.D. *: A star indicates statistical significance compared to control group.
1,25-(OH)2D3 activates MMP13 through the mitogen-activated protein kinase p38 pathway. (A) Fold change in RNA levels of MMP13. ATDC5 chondrocytes were cultured in DMEM and treated with 10 nM of 1,25-(OH)2D3 for 24 hr. SB203580 (10 μM) and SP600125 (20 μM) were added to the culture medium 2 hr before 1,25-(OH)2D3 treatment. RNA expression level of MMP13 was determined by quantitative real-time RT-PCR. Level of RNA from control group was normalized to a value of 1. (B) Inhibition by the p38 inhibitor SB203580 of 1,25-(OH)2D3-induced increase in MMP13 protein and phosphorylation of p38 in ATDC5 chondrocytes. Confluent chondrocytes were cultured and incubated with or without SB203580 for 2 hr before addition of 10 nM 1,25-(OH)2D3 for 24 hr. Total cell lysates were prepared and analyzed by Western blot using antibodies that recognize MMP13, phosphor-p38 and β-actin.
1,25-(OH)2D3 activates the MMP13 promoter in a dose-dependent manner. ATDC5 chondrocytes were transfected with a 1kb MMP13 promoter-luciferase reporter plasmid without or with increasing amounts of 1,25-(OH)2D3 as indicated. Twenty-four hours post-transfection, cell extracts were prepared and analyzed for luciferase activity. Luciferase activity was normalized by β-galactosidase activity. Values are presented as the mean ±S.D.
Osteoarthritis (OA) is the most prevalent degenerative joint disease. The highly regulated balance of matrix synthesis and degradation is disrupted in OA, leading to progressive breakdown of articular cartilage. The molecular events and pathways involved in chondrocyte disfunction of cartilage in OA are not fully understood. It is known that 1,25-dihydroxyvitamin D₃ (1,25-(OH)2D3) is synthesized by macrophages derived from synovial fluid of patients with inflammatory arthritis. Vitmain D receptor is expressed in chondrocytes within osteoarthritic cartilage, suggesting a contributory role of 1,25-(OH)2D3 in the aberrant behavior of chondrocytes in OA. However, the physiological function of 1,25-(OH)2D3 on chondrocytes in OA remains obscure. Effect of 1,25-(OH)2D3 on gene expression in chondrocytes was investigated in this study. We found that 1,25-(OH)2D3 activated MMP13 expression in a dose-dependent and time-dependent manner, a major enzyme that targets cartilage for degradation. Interestingly, a specific mitogen-activated protein kinase p38 inhibitor SB203580, but not JNK kinase inhibitor SP600125, abrogated 1,25-(OH)2D3 activation of MMP13 expression. 1,25-(OH)2D3-induced increase in MMP13 protein level was in parallel with the phosphorylation of p38 in chondrocytes. To further address the effect of 1,25-(OH)2D3 on MMP13 expression, transfection assays were used to show that 1,25-(OH)2D3 activated the MMP13 promoter reporter expression. MMP13 is known to target type II collagen and aggrecan for degradation, two major components of cartilage matrix. We observed that the treatment of 1,25-(OH)2D3 in chondrocytes results in downregulation of both type II collagen and aggrecan while MMP13 was upregulated. Taken together, we provide the first evidence to demonstrate that 1,25-(OH)2D3 activates MMP13 expression through p38 pathway in chondrocytes. Since MMP13 plays a major role in cartilage degradation in OA, we speculate that the ability of 1,25-(OH)2D3 to potentiate MMP13 expression might facilitate cartilage erosion at the site of inflammatory arthritis.
 
Protective role of Smad2 in renal fibrosis. Smad2 physically binds Smad3 but prevents Smad3 from activation through two possible mechanisms: 1) competitively inhibit Smad3 binding to the TβRI for phosphorylation; 2) block phosphorylated Smad3 nuclear translocation and binding to the DNA sequences, therefore inhibiting Smad3-mediated renal fibrosis. Blue lines (symbols) indicate protective or negative regulation pathways, while red arrows (symbols) represent pathogenic or positive regulation pathways.
Smad3-dependent miRNAs in renal fibrosis. TGF-β1 acts by stimulating Smad3 to positively regulate miR-21 and miR-192, but negatively regulate the miR-29 or miR-200 families, to mediate renal fibrosis. Blue lines (symbols) indicate protective or negative regulation pathways, while red arrows (symbols) represent pathogenic or positive regulation pathways.
TGF-β1 has been long considered as a key mediator in renal fibrosis and induces renal scarring largely by activating its downstream Smad signaling pathway. Interestingly, while mice overexpressing active TGF-β1 develop progressive renal injury, latent TGF-β1 plays a protective role in renal fibrosis and inflammation. Under disease conditions, Smad2 and Smad3 are highly activated, while Smad7 is degraded through the ubiquitin proteasome degradation mechanism. In addition to TGF-β1, many pathogenic mediators such as angiotensin II and advanced glycation end products can also activate the Smad pathway via both TGF-β-dependent and independent mechanisms. Smads interact with other signaling pathways, such as the MAPK and NF-κB pathways, to positively or negatively regulate renal inflammation and fibrosis. Studies from gene knockout mice demonstrate that TGF-β1 acts by stimulating its downstream Smads to diversely regulate kidney injury. In the context of renal fibrosis and inflammation, Smad3 is pathogenic, while Smad2 and Smad7 are protective. Smad4 exerts its diverse roles by transcriptionally enhancing Smad3-mediated renal fibrosis while inhibiting NF-κB-driven renal inflammation via a Smad7-dependent mechanism. Furthermore, we also demonstrated that TGF-β1 acts by stimulating Smad3 to positively or negatively regulate microRNAs to exert its fibrotic role in kidney disease. In conclusion, TGF-β/Smad signaling is a major pathway leading to kidney disease. Smad3 is a key mediator in renal fibrosis and inflammation, whereas Smad2 and Smad7 are renoprotective. Smad4 exerts its diverse role in promoting renal fibrosis while inhibiting inflammation. Thus, targeting the downstream TGF-β/Smad3 signaling pathway by gene transfer of either Smad7 or Smad3-dependent microRNAs may represent a specific and effective therapeutic strategy for kidney disease.
 
Differential expression of versican and its isoforms. (A) Q-RT-PCR analysis of the expression of the versican mRNA isoforms in the gastric carcinoma tissue and the adjacent normal tissue samples. Four isoforms were analysed in the gastric carcinoma tissue and adjacent normal tissue. V1 revealed a significantly higher expression (*P<0.05). (B) RT-PCR analysis of the versican isoforms. The results agree well with those of the Q-RT-PCR analysis shown in Fig. 1 A. (C) The expression of versican V0/V1 was determined by Western blot. Versican was significantly up-regulated in gastric carcinoma tissue than in adjacent normal tissue (*P<0.05). GAPDH was used as the loading control. (D) Versican isoform variants in human gastric carcinoma cell lines. V0 and V1 were found to be expressed in most gastric cells. GAPDH was used to adjust for cDNA quantity.
Effects of exogenous IL-11 on the expression of the versican V0 and V1 isoforms in AGS and MKN45 cells. (A) Four different concentrations (10, 50, 100, and 200 ng/ml) of rhIL-11 were added to AGS and MKN45 cells. In AGS cells, V0 and V1 were both upregulated in a concentration-dependent manner. However, in MKN45 cells treated with 100 ng/ml of rhIL-11, V0 and V1 reached peak expression levels of 2.6-fold and 2.3-fold, respectively (*P<0.05, **P<0.01). (B) After treatment of AGS and MKN45 cells with rhIL-11 (100 ng/ml) at three different time points (hours 24, 48 and 72), V0 and V1 were both upregulated in a time-dependent manner (*P<0.05, **P<0.01). The quantity of each target was normalised against the quantity of GAPDH. Mean values of the control group were set to a value of 1. n=3. Results are given as the mean±SD.
Effect of exogenous IL-11 on the migration and proliferation of gatric carcinoma cell lines. (A) The directed migratory capacities of AGS cells treated with rhIL-11 were evaluated using a Transwell migration study. Sample images showing the pro-migratory effect of IL-11 on AGS cells after staining the transmigrated cells with crystal violet (magnification, ×100). Exogenous IL-11 induced migration more significantly than the untreated control and BSA did. (B) Graphs indicate the average number of cells from five random high-power fields in the indicated groups. Data are presented as mean±SD based on three independent experiments (**P<0.01). (C) Proliferative effects of rhIL-11 in gatric carcinoma cell lines (AGS and MKN45 cells) were measured by CCK-8 assay. Both cell lines exhibited significantly growth until 60 hours (*P<0.05, **P<0.01). (D) A scratch migration assay was used to assess the migration of AGS and MKN45 cells that were treated with rhIL-11 (100 ng/ml). The migration rate was quantified by measuring the distance between the edges of the scratch at t=0 relative to t=36 h. After incubation, the cells can be seen migrating into the wound with a decreased distance between the edges.
(A): Q-RT-PCR was performed to validate versican V0 and V1 inhibition after AGS cells were transfected with siRNA-versican. (B) Western blot analysis of versican V0/V1 and IL-11 protein levels. The results in (A) and (B) showed that versican V0/V1 were significantly inhibited by siRNA-V0/V1 but not by the negative control siRNA in AGS cells, and the expression of endogenous IL-11 in cells was not affected by both siRNAs. (C) The cell viability of siRNAs transfected was measured by CCK-8 assay. In both 24 and 48h time points, the cell viability didn't show a significantly difference between siRNAs treated and untreated control groups (P>0.05). (D) The effect of versican on AGS cell migration mediated by rhIL-11 was analysed in a transwell assay as described in the methods section. rhIL-11-treated cells showed significantly higher migration rates (**P<0.01). siRNA-versican was able to decrease migration, both with and without rhIL-11, compared to the untreated control and BSA (**P<0.01). (E) The migrated cells were quantified and expressed as the mean±SD of three independent experiments.
Versican, a ubiquitous component of the extracellular matrix (ECM), accumulates both in tumor stroma and cancer cells and is highly regulated by various cytokines. The aberrant expression of versican and its isoforms is known to modulate cell proliferation, differentiation, and migration, all of which are features of the invasion and metastasis of cancer; versican is also known to favour the homeostasis of the ECM. Interleukin-11 (IL-11) is an important cytokine that exhibits a wide variety of biological effects in gastric cancer development. Here, we analysed the expression of versican isoforms and found that the major isoforms expressed by both gastric carcinoma tissue and gastric cell lines were V0 and V1, and V1 was significantly higher in gastric carcinoma tissue. The treatment of the gastric cell lines AGS and MKN45 with rhIL-11 resulted in a significant increase in the expression of V0 and V1. Exogenous IL-11 increased migration in AGS and MKN45 cells, whereas these effects were reversed when the expression of V0 and V1 were abolished by siRNA targeting versican V0/V1. Collectively, these findings suggest that the abnormally expressed versican and its isoforms participate, at least in part, in the progress of gastric carcinoma triggered by IL-11.
 
Mutated p53 gene is related to the instability of cell growth and cell cycle progression. We aimed to evaluate the association between endometriosis and p53 codon 11, 72 and 248 gene polymorphisms. Women were divided into two groups: (1) moderate/severe endometriosis (n=148), and (2) non-endometriosis groups (n=150). P53 gene polymorphisms include codon11 Glu/Gln or Lys (GAG->CAG or AAG), codon 72 Arg/Pro (CGC->CCC), and codon 248 Arg/Thr (CGG->TCG). These gene polymorphisms were amplified by polymerase chain reaction and detected by electrophoresis after restriction enzyme (Taq I, BstU I, Hap II) digestions. Associations between the endometriosis and p53 polymorphisms were evaluated. The distributions of p53 codon 72 polymorphisms in both groups were significantly different. The proportions of Arg homozygotes/heterozygotes/Pro homozygotes in both groups were 9.5/66.2/24.3% and 30.7/50/19.3%. The proportions of Arg/Pro alleles were 42.6/57.4% and 56/44%. The distributions of p53 codon 11 and 248 polymorphisms in both groups were non-significantly different. All individuals appeared the wild genotypes (Glu11 and Arg248 homozygotes). Association between endometriosis and p53 codon 72 polymorphism exists. P53 codon 72*Pro-related genotype and allele are related with higher susceptibility of endometriosis. P53 codon 11 and 248 polymorphisms are not related with endometriosis susceptibility.
 
The clinical characteristics of hepatocellular carcinoma (HCC) cases and controls.
Primers and PCR-RFLP analysis for genotyping XRCC1 gene polymorphisms.
The genotypic and allelic frequencies of XRCC1 polymorphisms in case and groups.
Association between hepatocellular carcinoma (HCC) risk and XRCC1 gene polymorphisms.
The human X-ray repair complementing group 1 gene (XRCC1) is an important candidate gene influencing hepatocellular carcinoma (HCC) susceptibility. The objective of this study was to detect the association between c.1161G>A and c.1779C>G variants of XRCC1 gene and HCC risk. This study was conducted in Chinese population consisting of 623 HCC cases and 639 controls. These two genetic variants could be genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The association of XRCC1 gene variants with the risk of HCC was investigated under different genetic models. Our findings suggested that the genotypes/alleles from c.1161G>A and c.1779C>G genetic variants were statistically associated with HCC risk. As for the c.1161G>A, the AA genotype was statistically associated with the increased risk of HCC compared to GG wild genotype (OR = 2.36, 95% CI 1.63-3.40, P < 0.001). As for the c.1779C>G, the risk of HCC was significantly higher for GG genotype compared to CC wild genotype (OR = 2.17, 95% CI 1.51-3.12, P < 0.001). Furthermore, significant differences in the risk of HCC were also detected in other genetic models for these two variants. The allele-A of c.1161G>A and allele-G of c.1779C>G variants may contribute to the susceptibility of HCC (A versus G: OR = 1.48, 95% CI 1.26-1.75, P < 0.001 and G versus C: OR = 1.51, 95% CI 1.28-1.78, P < 0.001). Our data indicated that these two variants of XRCC1 gene were statistically associated with HCC risk in Chinese population.
 
Many viruses are known to undergo rapid evolutionary changes under selective pressures. The HIV-1 envelope glycoprotein 120 (gp120) shows extreme selection for NXS/T sequons, the potential sites of N-glycosylation. Although the average number of sequons in gp120 appears to be relatively stable in the recent past, even slight changes in the distribution of sequons may potentially play crucial roles in protein interaction and viral infection. This study tracked the prevalence and distribution of NXS/T sequons in gp120 over a period of 29 years (from 1981 to 2009). The gp120 showed location specific distribution of sequons with higher density in the outer domain of the molecule. The NXT sequon density decreased in the outer domain (despite the increase in the sequon specific amino acid threonine), but increased in the inner domain. By contrast, the NXS sequon density increased specifically in the outer domain. Related changes were also seen in the distribution probabilities of sequons in two domains. The results indicate that the gp120, chiefly in subtype B, is redistributing NXS/T sequons within the molecule with specific selection for NXS sequons. The subtle evolution of sequons in gp120 may have implications in viral resistance and infection.
 
Circulating miRNAs have been shown as promising biomarkers for various pathologic conditions. The aim of this study was to clarify that circulating miR-1 and miR-126 in human plasma might be useful as biomarkers in acute myocardial infarction (AMI). In our study, after pre-test, two candidate miRNAs were detected by using real-time RT-PCR. Cardiac troponin I (cTnI) concentrations were measured by ELISA assay in plasma from patients with AMI (n=17) and healthy subjects (n=25), simultaneously. Increased miR-1 and decreased miR-126 in plasma from patients with AMI after the onset of symptoms compared with healthy subjects were found. A remarkable finding in this study is that miR-1, miR-126 and cTnI expression levels exhibited the same trend. Our results suggest that the plasma concentrations of miR-1 and miR-126 may be useful indicators for AMI.
 
Effect of azithromycin (AZM) on RAW264.7cell growth and cell viability. Cells were cultured with alamarBlue dye solution in absence or presence of AZM (0.1, 1.0, 10, and 20 μg/ml) for 72 h, and the optical density (OD) at 595 nm was measured at the indicated time points. Proliferation and viability of RAW264.7 cells were determined in relation to growth curve, based on the measurement of OD at each time point. Data are mean value of six independent experiments.
Azithromycin (AZM), a 15-member macrolide antibiotic, possesses anti-inflammatory activity. Macrophages are important in innate and acquired immunity, and produce pro-inflammatory cytokines such as interleukin (IL)-12, which are composed of subunit p40 and p35. The key function of IL-12 is the induction and maintenance of T-helper-1 responses, which is associated with the pathogenesis of chronic inflammatory diseases. We investigated the effect of azithromycin on IL-12p40 production in macrophages after lipopolysaccharide (LPS)/interferon (IFN)-gamma stimulation. RAW264.7 macrophage cell line was pre-treated with vehicle or AZM, followed by the stimulation with LPS/IFN-gamma. We measured IL-12 production by RT-PCR and ELISA. IL-12 transcriptional regulation was assessed by electrophoretic mobility shift assay and reporter assay. Phosphorylation of activator protein (AP)-1 and interferon consensus sequence binding protein (ICSBP) was assessed by immunoprecipitation using phosphotyrosine antibody, and immunoblotting using specific antibodies against JunB and ICSBP. AZM reduced the induction of IL-12p40 by LPS/IFN-gamma in a dose dependent manner. AZM inhibited the binding of AP-1, nuclear factor of activated T cells (NFAT), and ICSBP, to the DNA binding site in the IL-12p40 promoter. AZM also reduced LPS/IFN-gamma-induced IL-12p40 promoter activity. Phosphorylation of JunB and ICSBP was inhibited by azithromycin-treatment in stimulated cells. In conclusion, AZM reduced IL-12p40 transcriptional activity by inhibiting the binding of AP-1, NFAT, and ICSBP to the promoter site. This may represent an important mechanism for regulating the anti-inflammatory effects of AZM in macrophages.
 
Endogenous BMP-13 gene expression in undifferentiated BM MSCs by real-time PCR analysis. BMP-13 gene expression was detected at days 1, 3, 5 and 7 in MSC expansion cultures and compared to that of BMP-2 and -7 in parallel. Relative expression was calculated as a ratio to the average value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT1). The individual BMP-2 and -7 time-course expression levels are shown in insets.  
Bone morphogenetic protein-13 (BMP-13) plays an important role in skeletal development. In the light of a recent report that mutations in the BMP-13 gene are associated with spine vertebral fusion in Klippel-Feil syndrome, we hypothesized that BMP-13 signaling is crucial for regulating embryonic endochondral ossification. In this study, we found that BMP-13 inhibited the osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. The endogenous BMP-13 gene expression in MSCs was examined under expansion conditions. The MSCs were then induced to differentiate into osteoblasts in osteo-inductive medium containing exogenous BMP-13. Gene expression was analysed by real-time PCR. Alkaline phosphatase (ALP) expression and activity, proteoglycan (PG) synthesis and matrix mineralization were assessed by cytological staining or ALP assay. Results showed that endogenous BMP-13 mRNA expression was higher than BMP-2 or -7 during MSC growth. BMP-13 supplementation strongly inhibited matrix mineralization and ALP activity of osteogenic differentiated MSCs, yet increased PG synthesis under the same conditions. In conclusion, BMP-13 inhibited osteogenic differentiation of MSCs, implying that functional mutations or deficiency of BMP-13 may allow excess bone formation. Our finding provides an insight into the molecular mechanisms and the therapeutic potential of BMP-13 in restricting pathological bone formation.
 
CtBP1 is a predicted target for miR-137. (a) Diagram of CtBP1 mRNA depicting the predicted miR-137 site in the 3'UTR region. (b) CtBP1 protein levels in A375 and WM278 cells. Tubulin was used as a loading control. (c) miR-137 levels of the A375 and WM278 cells. miR-137 level was determined by the Taqman assay. Data were normalized to the 18s mRNA. Samples were assayed in triplicate for each experiment and at least two independent experiments were performed. Data are presented as mean ± SEM from a representative experiment. (d) Conservation of the miR-137 site in CtBP1 in different species.
Carboxyl-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor that represses expression of various tumor suppressor genes. In the present study, we identified miR-137 as a potential regulator of CtBP1 expression in melanoma cells. Expression of miR-137 in melanoma cell lines was found to inversely correlate with CtBP1 levels. Target Scan predicted a putative site for miR-137 within the CtBP1 3' untranslated region (3'UTR) at nt 710-716, which is highly conserved across species. To explore the mechanism of miR-137 targeting CtBP1, we performed an Argonaute 2 (Ago2)-pull down assay, and miR-137 was identified in complex with CtBP1 mRNA. miR-137 suppressed CtBP1 3' UTR luciferase-reporter activity, and this effect was lost with deletion of the putative 3' UTR target-site. Consistent with the results of the reporter assay, ectopic expression of miR-137 reduced expression levels of CtBP1. Furthermore, expression of miR-137 increased the immediate downstream effectors of CtBP1, such as E-cadherin and Bax. The human miR-137 gene is located at chromosome 1p22, which has previously been determined to be a susceptive region for melanoma. This study suggests miR-137 may act as a tumor suppressor by directly targeting CtBP1 to inhibit epithelial-mesenchymal transition (EMT) and inducing apoptosis of melanoma cells, thus illustrating a functional link between miR-137 and CtBP1 in melanoma development.
 
The Hox gene cluster has been a key paradigm for a generation of developmental and evolutionary biologists. Since its discovery in the mid-1980's, the identification, genomic organization, expression, colinearity, and regulation of Hox genes have been immediate targets for study in any new model organism, and metazoan genome projects always refer to the structure of the particular Hox cluster(s). Since the early 1990's, it has been dogma that vertebrate Hox clusters are composed of thirteen paralogous groups. Nonetheless, we showed that in the otherwise prototypical cephalochordate amphioxus (Branchiostoma floridae), the Hox cluster contains a fourteenth Hox gene, and very recently, a 14(th) Hox paralogous group has been found in the coelacanth and the horn shark, suggesting that the amphioxus cluster was anticipating the finding of Hox 14 in some vertebrate lineages. In view of the pivotal place that amphioxus occupies in vertebrate evolution, we thought it of considerable interest to establish the limits of its Hox gene cluster, namely resolution of whether more Hox genes are present in the amphioxus cluster (e.g., Hox 15). Using two strategies, here we report the completion and characterization of the Hox gene content of the single amphioxus Hox cluster, which encompasses 650 kb from Hox1 to Evx. Our data have important implications for the primordial Hox gene cluster of chordates: the prototypical nature of the single amphioxus Hox cluster makes it unlikely that additional paralogous groups will be found in any chordate lineage. We suggest that 14 is the end.
 
QTL reported on BTA14 for milk production traits in dairy cattle.
QTL reported on BTA14 for growth, carcass, meat quality and eating quality traits in beef cattle.
QTL reported on BTA14 for health, reproduction and udder related traits in dairy cattle.
Bovine chromosome 14 (BTA14) has been widely explored for quantitative trait loci (QTL) and genes related to economically important traits in both dairy and beef cattle. We reviewed more than 40 investigations and anchored 126 QTL to the current genome assembly (Btau 4_0). Using this anchored QTL map, we observed that, in dairy cattle, the region spanning 0 - 10 Mb on BTA14 has the highest density QTL map with a total of 56 QTL, mainly for milk production traits. It is very likely that both somatic cell score (SCS) and clinical mastitis share some common QTL in two regions: 61.48 Mb - 73.84 Mb and 7.86 Mb - 39.55 Mb, respectively. As well, both ovulation rate and twinning rate might share a common QTL region from 34.16 Mb to 65.38 Mb. However, there are no common QTL locations in three pregnancy related phenotypes: non-return rate, pregnancy rate and daughter pregnancy rate. In beef cattle, the majority of QTL are located in a broad region of 15 Mb - 45 Mb on the chromosome. Functional genes, such as CRH, CYP11B1, DGAT1, FABP4 and TG, as potential candidates for some of these QTL, were also reviewed. Therefore, our review provides a standardized QTL map anchored within the current genome assembly, which would enhance the process of selecting positional and physiological candidate genes for many important traits in cattle.
 
Recently we demonstrated that IGF-1 expression is increased in the diabetic kidney and that it may involve in renal hypertrophy and extracellular matrix protein (ECM) accumulation in mesangial cells as seen in diabetic glomerulopathy. The present study investigates the molecular mechanism(s) of IGF-1 and Akt/glycogen synthase kinase-3beta (GSK-3beta) signaling pathway in the regulation of fibronectin and cyclin D1 expression and survival of renal mesangial cells. A proteomic approach is also employed to identify protein targets of IGF-1 signaling via GSK-3beta inhibition in mesangial cells. We show that IGF-1 (100 ng/ml) significantly increases the protein kinase Akt/PKB activity (1.5-2-fold, p<0.05) within 1-5 minutes, which is completely blocked by the presence of 100 nM Wortmannin (phosphatidyl-inositol 3-kinase inhibitor). Akt activation is coupled with Ser9 phosphorylation and inactivation of its down-stream target GSK-3beta. IGF-1 increases the cyclic AMP-responsive element (CRE) binding transcription factor CREB phosphorylation at Ser 133 and CRE-binding activity in mesangial cells, which parallels cyclin D1 and fibronectin expressions. Both proteins are known to have CRE-sequences in their promoter regions upstream of the transcription start site. Suppression of GSK-3beta by SB216763 (100 nM) increases CREB phosphorylation, cyclin D1 and fibronectin levels. Two dimensional gel electrophoresis followed by MALDI-TOF mass spectrometric analysis of mesangial proteins reveals that IGF-1 treatment or an inhibition of GSK-3beta increases the expression of the phosphorylated Ser/Thr binding signal adapter protein 14-3-3zeta. Immuno-precipitation of 14-3-3zeta followed by Western blotting validates the association of phosphorylated GSK-3beta with 14-3-3zeta in renal mesangial cells. Stable expression of a constitutively active GSK-3beta(Ser9Ala) induces cell death while overexpression of HA-tagged 14-3-3zeta increases cell viability as measured by MTT assays. These results indicate that the Akt/GSK-3beta pathway and the adapter protein 14-3-3zeta may play an important role in IGF-1 signaling and survival of mesangial cells in diabetic nephropathy.
 
Background: MicroRNAs (miRNAs) play important roles in many biological processes, including cancer development. Among those miRNAs, miR-143 shows tumor-suppressive activity in some human cancers. However, the function and mechanism of miR-143 in lung cancer cells remains unknown. Here we explored the role of miR-143 in lung cancer. Results: According to qRT-PCR, we found that miR-143 was notably down-regulated in 19 NSCLC tissues and 5 cell lines. In vitro experiments showed us that miR-143 could significantly suppress the migration and invasion of NSCLC cell lines while it had no effects on the growth of NSCLC cell lines, and in vivo metastasis assay showed the same results. Finally, we found that the mechanism of miR-143 inhibiting the migration and invasion of NSCLC might be through targeting CD44v3. Conclusions: The up-regulated miR-143 in lung cancer could significantly inhibit cell migration and invasion, and this might work through targeting CD44v3, which was newly identified by us.
 
Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.
 
SIRT1 has been considered as a tumor promoter because of its increased expression in some types of cancers and its role in inactivating proteins that are involved in tumor suppression and DNA damage repair. However, recent studies demonstrated that SIRT1 levels are reduced in some other types of cancers, and that SIRT1 deficiency results in genetic instability and tumorigenesis, while overexpression of SIRT1 attenuates cancer formation in mice heterozygous for tumor suppressor p53 or APC. Here, I review these recent findings and discuss the possibility that activation of SIRT1 both extends lifespan and inhibits cancer formation.
 
Tissue-specific and cell-specific expression pattern of miR-148a in mouse (A), miR-148a expression was detected in various tissues of mouse by Northern blot. (B), ISH analysis showed that miR-148a expression was located at bottom half of gastric epithelium in mouse. Original magnification, ×200.
miR-148a promoted cell proliferation in gastric cancer cells. (A), miR-148a expression was measured by Northern blot in 6 human gastric cancer cell lines. (B), miR-148a downregulation with AS was confirmed by Northern Blot in a representative transfectant. (C), miR-148a downregulation inhibited cell proliferation in AGS, BGC-823 and SGC-7901 cells. Gastric cancer cells were transfected with miR-148a AS or scrambled miRNA AS (as a negative-control, NC AS). Cell viability was determined by cell count 72 and 96 hours after transfection. The numbers of viable cells transfected with NC AS were set at 1. (D), miR-148a promoted cell proliferation in AGS cells. miR-148a overexpression with mimic were confirmed by Northern Blot in AGS cells. Cell viability was determined by cell count 72 and 96 hours after transfection. (E), Northern blot revealed that miR-148a expression was increased in stably miR-148a-expressing AGS cells (miR-148a) compared with AGS stably transfected with empty vector (vector). Cell count showed that the cell numbers at 48 and 72 hours were increased in stably miR-148a-expressing AGS cells. (F), Cell cycle analysis showed that AGS cells transiently transfected with miR-148a mimic exhibited increased population of cells at S phase 24 hours after transfection. (G), Stably miR-148a-expressing AGS cells (AGS-miR-148a) also showed increased population of cells at S phase. (H), FCAS analysis showed no obvious change was observed in apoptosis induced by Etoposide. Values represent mean ± SD. n=3. *P<0.05, **P<0.01.
miR-148a was frequently and dramatically downregulated in human advanced gastric cancer and showed an inverse correlation with p27 expression in tumor samples. (A), miR-148a expression was determined by Northern blot in 10 advanced gastric primary tumors, each one paired with adjacent nontumor gastric tissue from the same patient. (B), Statistically significant downregulation of miR-148a in gastric cancers over matching normal tissues. P=0.005 by Wilcoxon signed rank test. Bars represent mean. (C), p27 expression was examined by Western blot in the same samples. (D), The relationship between miR-148a and p27 expression was detected by Spearman rank correlation analysis in tumor samples. r=0.648. p<0.05.
Accumulating evidence has shown that miRNAs are aberrantly expressed in human gastric cancer and crucial to tumorigenesis. Herein, we identified the role of miR-148a in gastric cell proliferation. miR-148a knockdown inhibited cell proliferation in gastric cancer cell lines. Conversely, miR-148a overexpression promoted cell proliferation and cell cycle progression. p27, a key inhibitor of cell cycle, was verified as the target of miR-148a, indicating miR-148a might downregulate p27 expression to promote gastric cell proliferation. Moreover, we confirmed that miR-148a expression was frequently and dramatically downregulated in human advanced gastric cancer tissues, and observed a good inverse correlation between miR-148a and p27 expression in tumor samples. Thus, our results demonstrated that miR-148a downregulation might exert some sort of antagonistic function in cell proliferation, rather than promote cell proliferation in gastric cancer.
 
The exon ⁄ intron organizations of porcine OLFML3 gene.
Deduced amino acid sequence of porcine OLFML3 compared with mouse OLFML3 (GenBank accession no. NP_598620.2) and human OLFML3 (GenBank accession no. NP_064575.1). Shading shows identical amino acid residues among the three species. Common structural domains are indicated by boxes including coiled coil region and Olfactomedin-like domains.
Phylogenetic tree and domain of porcine OLFML3 protein. (a) Phylogenetic tree of OLFML3 in different species. The GenBank accession numbers of those sequences are as follows: human: NP_064575.1, cattle: NP_001068665.1, rat: NP_001101178.1, mouse: NP_598620.2, Gallus: NP_001035107.1. The porcine OLFML3 amino acid sequence was deduced from the full cDNA sequence in this study. (b) Domain of porcine OLFML3 protein. The porcine OLFML3 precursor protein contains coiled coil region and Olfactomedin-like domain.
OLFML3 3'UTR has conserved miR-155 target sites. (a) OLFML3 was predicted as a target of miR-155 using the PicTar. (b) OLFML3 was predicted as a target of miR-155 using TargetScan program (Version 5.1). (c) The complementary site for OLFML3 and miR-155 was mapped using DNAMAN. The upper part represents OLFML3 and the lower part represents miR-155. The corresponding plot shows conservation across species.
OLFML3 is a primary target for miR-155. a. Schematic representation of the construct used in the luciferase assay. A 485-bp region of the porcine OLFML3 3'UTR containing one putative miR-155 target site was cloned into the psiCHECK-2 vector. b. Overexpression of porcine miR-155 in PK15 and PIEC cells. Histogram indicates overexpression of miR-155 after transfection for 48 h. Mature miR-155 was analyzed by real-time PCR. c. miR-155:OLFML3 3'UTR duplex. The sequence marked in red is OLFML3 3'UTR, and the sequence marked in green is the seed sequence of miR-155 in human. d. Cotransfection of porcine pre-miR-155 or control and porcine OLFML3 3'UTR-derived psiCHECK-2 construct or mutant in PK15 and PIEC cells. Renilla activity at 48 h post-transfection shows a significant decrease in normalized values than in control and mutant in both cell types. Five replicates were performed for each group and the experiment was repeated at least three times. ** indicates a p-value of less than 0.01 in a Student's t-test.
The Olfactomedin-like 3 (OLFML3) gene has matrix-related function involved in embryonic development. MicroRNA-155 (miR-155), 21- to 23-nucleotides (nt) noncoding RNA, regulated myogenesis by target mRNA. Our LongSAGE analysis suggested that OLFML3 gene was differently expressed during muscle development in pig. In this study, we cloned the porcine OLFML3 gene and detected its tissues distribution in adult Tongcheng pigs and dynamical expression in developmental skeletal muscle (12 prenatal and 10 postnatal stages) from Landrace (lean-type) and Tongcheng (obese-type) pigs. Subsequently, we analyzed the interaction between OLFML3 and miR-155. The OLFML3 was abundantly expressed in liver and pancreas, moderately in lung, small intestine and placenta, and weakly in other tissues and postnatal muscle. There were different dynamical expression patterns between Landrace and Tongcheng pigs during prenatal skeletal muscle development. The OLFML3 was down-regulated (33-50 days post coitus, dpc), subsequently up-regulated (50-70 dpc), and then down-regulated (70-100 dpc) in Landrace pigs, while in Tongcheng pigs, it was down-regulated (33-50 dpc), subsequently up-regulated (50-55 dpc) and then down-regulated (55-100 dpc). There was higher expression in Tongcheng than Landrace in prenatal muscle from 33 to 60 dpc, and opposite situation from 65 to 100 dpc. Dual luciferase assay and real time PCR documented that OLFML3 expression was regulated by miR-155 at mRNA level. Our research indicated that OLFML3 gene may affect prenatal skeletal muscle development and was regulated by miR-155. These finding will help understanding biological function and expression regulation of OLFML3 gene in mammal animals.
 
Overexpression of SIRT3 induced MnSOD expression. (A) A MnSOD promoter luciferase reporter plasmid (pMnSOD-Luc) was transfected into wt-SIRT3, mt-SIRT, and vector control cell lines. Luciferase activity was normalized to β-galactosidase activity and presented as luciferase/β-galactosidase relative units. All results are the mean of at least three separate experiments. Error bars around data points represent one standard deviation about the arithmetic mean. (B) pMnSOD-Luc was co-transfected into HCT116 cells with either pCMV-SIRT3-wt or pCMV-SIRT3-mt expression vectors or luciferase assays were done as described above. * Indicates P < 0.05 by t-test.
Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.
 
In this study, we characterized the intratumoral expression of IL-17 and CD8(+) TILs in gastric adenocarcinoma patients after resection and determined the correlation between the survival probability of gastric adenocarcinoma patients and the expression of IL-17 in tumor. Expression of IL-17 and CD8 was assessed by immunohistochemistry, and the prognostic effects of intratumoral IL-17 expression and CD8(+) TILs were evaluated by Cox regression and Kaplan-Meier analysis. Immunohistochemical detection revealed the presence of IL-17 and CD8(+) cells in gastric adenocarcinoma tissue samples (90.6%, 174 out of 192 patients and 96.9%, 186 out of 192 patients, respectively). We have also found that intratumoral IL-17 expression was significantly correlated with age (p=0.004) and that the number of CD8(+)TILs was significantly correlated with UICC staging (p=0.012) and the depth of tumor invasion (p=0.022). The five-year overall survival probability among patients intratumorally expressing higher levels of IL-17 was significantly better than those expressing lower levels of IL-17 (p=0.036). Multivariate Cox proportional hazard analyses revealed that intratumoral IL-17 expression (HR: 0.521; 95% CI: 0.329-0.823; p=0.005) was an independent factor affecting the five-year overall survival probability. We conclude that low levels of intratumoral IL-17 expression may indicate poor prognosis in gastric adenocarcinoma patients.
 
Glioblastoma multiforme (GBM) is the most common form of malignant glioma, characterized by genetic instability and unpredictable clinical behavior. GBM is marked by an extremely poor prognosis with median overall survival of 12~14 months. In this study, we detected the CD137L-expressing cells and IL-17-expressing cells in tumor tissues resected from patients with GBM. Expression of CD137L and IL-17 were assessed by immunohistochemistry, and the prognostic value of CD137L and IL-17 expression within the tumor tissues were assessed by Cox regression and Kaplan-Meier analysis. Immunohistochemical detection showed that positive cells of CD137L and IL-17 in glioblastoma tissue samples were 46.3% (19/ 41) and 73.2% (30/41) respectively. Expression of CD137L was not correlated with overall survival of GBM patients (P=0.594), while significantly longer survival rate was seen in patients with high expression of IL-17, compared to those with low expression of IL-17 (P=0.007). In addition, we also found that IL-17 expression was significantly correlated with Progression-free survival (PFS) (P=0.016) and death rate (P=0.01). Furthermore, multivariate Cox proportional hazard analyses revealed that IL-17 (P=0.018) and PFS (P=0.028) were independent factors affecting the overall survival probability. Kaplan-Meier analysis showed that PFS of high expression of IL-17 group were significantly longer (P=0.004) than low expression group with GBM. It is concluded that high levels of IL-17 expression in the tumor tissues may be a good prognostic marker for patients with GBM.
 
Quantitative or complex traits are determined by the combined effects of many loci, and are affected by genetic networks or molecular pathways. In the present study, we genotyped a total of 138 mutations, mainly single nucleotide polymorphisms derived from 71 functional genes on a Wagyu x Limousin reference population. Two hundred forty six F(2) animals were measured for 5 carcass, 6 eating quality and 8 fatty acid composition traits. A total of 2,280 single marker-trait association runs with 120 tagged mutations selected based on the HAPLOVIEW analysis revealed 144 significant associations (P < 0.05), but 50 of them were removed from the analysis due to the small number of animals (< or = 9) in one genotype group or absence of one genotype among three genotypes. The remaining 94 single-trait associations were then placed into three groups of quantitative trait modes (QTMs) with additive, dominant and overdominant effects. All significant markers and their QTMs associated with each of these 19 traits were involved in a linear regression model analysis, which confirmed single-gene associations for 4 traits, but revealed two-gene networks for 8 traits and three-gene networks for 5 traits. Such genetic networks involving both genotypes and QTMs resulted in high correlations between predicted and actual values of performance, thus providing evidence that the classical Mendelian principles of inheritance can be applied in understanding genetic complexity of complex phenotypes. Our present study also indicated that carcass, eating quality and fatty acid composition traits rarely share genetic networks. Therefore, marker-assisted selection for improvement of one category of these traits would not interfere with improvement of another.
 
Schematic representation of the 248-kb mitochondrial sequence on chromosome 12 and the corresponding sequences on a master circle of rice mitochondrial genome. (A) Three contiguous BAC clones carrying mitochondrial sequences are indicated by bold lines. cM, centimorgans. A black box and a circle represent a mitochondrial sequence and a centromere on chromosome 12, respectively. Four striped boxes show retrotransposon sequences. Three white boxes indicate sequences of unknown origin. (B) A presumptive mitochondrial master circle of the rice mitochondrial genome. Sequences with strong similarity are shown in the same colors
A model for the evolution of the 248-kb numtDNA on rice chromosome 12. Step 1, integration of a 190-kb mitochondrial fragment into chromosome 12. Step 2-1, integration of four retrotransposons into the 190-kb mitochondrial fragment. Step 2-2, recombination events within the mitochondrial genome
A 190-kb mitochondrial DNA sequence interrupted by seven foreign DNA segments was identified in rice chromosome 12. This fragment is the largest mitochondrial fragment translocated into the rice nuclear genome. The sequence is composed of a 190-kb segment of mitochondrial origin corresponding to 38.79% of the mitochondrial genome, 45 kb comprising four segments of retrotransposon origin, and 13 kb comprising three segments of unknown origin. The 190-kb sequence shows more than 99.68% similarity to the current mitochondrial sequence, suggesting that its integration into the nucleus was quite recent. Several sequences in the 190-kb segment have been rearranged relative to the current mitochondrial sequence, suggesting that the past and present arrangements of the mitochondrial genome differ. The four retrotransposons show no mutual sequence similarity and are integrated into different locations, suggesting that their integration events were independent, frequent, and quite recent. A fragment of the mitochondrial genome present in the nuclear genome, such as the 248-kb sequence characterized in this study, is a good relic with which to investigate the past mitochondrial genome structure and the behavior of independent retrotransposons during evolution.
 
The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.
 
Deletion of Bmpr1a leads to a lack of chondrogenesis at age of 2-weeks (right panels). (a) Representative BrdU stained images of the Bmpr1a cKO (conditional knockout) mice (crossing between Bmpr1a flox and aggrecan Cre ER with one time tamoxifen injection at newborn and harvesting at age of 2-weeks, right ) and the Cre negative littermate ( left ) showed no visible BrdU positive cells in the null growth plate; and (b-d). The safranin O stained images revealed lacks of hypertrophic chondrocytes and chondrocyte columns (b-c); and in the cKO metaphyses there were no trabeculae but a few large amounts of well-formed bone clusters, in which there was no signs of safranin O stains (the red color reflecting glycoproteins) in the cKO bone. 
Postnatal long bone growth is arrested in the 1-mon-old Bmpr1a cKO mice (right panels) after a one-time tamoxifen injection at birth. (a) Representative photographic and μ CT images showed the arrest of long bone growth in the Bmpr1a cKO mice with few signs of bone in the cKO metaphysis (upper panel); (b) The quantitative data revealed a significant reduction of femur length, trabecular number and bone volume in the cKO metaphysis; (c-d) The representative backscattered SEM (c) and FITC (d) images showed a lack of chondrocyte columns and trabeculae in the cKO femur. Instead, there were numerous spherical mineral bodies adjacent to a few large clusters of mature bones, in which the osteocytes (Ocy) were well-formed; (e) The H&E stain displayed similar bone clusters in the cKO metaphysis, which appeared from the surrounding mesenchymal cells; (f) The toluidine blue stain images showed no signs of the cartilage residues in the metaphysis bones that are directly formed from the mesenchymal cells (red half-circle arrows) compared to the control (red arrows, left); (g) The safranine O stain images revealed no cartilage extracellular matrices in the cKO bone compared to the control (yellow arrows); and (h) The polarized images disclosed numerous collagen fibers in the cKO growth plate compared to the age-matched control, in which there was no type I collagen. 
Removal of Bmpr1a leads to lacks of bone growth at newborns by one-time tamoxifen injection. Representative radiograph images showed a stop in lone bone growth at the following stages: 2-weeks, 1-month, 2-months and 5-months. The minor difference among different null long bone could be due to the variations of tamoxifen injections, as the injections occurred at newborn and some solution injected could not be leaked out. 
The lack of chondrogenic markers in the Bmpr1a null (cKO) growth plate (a-g) IHC stain images in the cKO growth plate indicated either a lack of signals of SOX9 (a) and IHH (b) separately, or faint signals of aggrecan (c), or ectopic expressions of OSX (d), DMP1 (e), periostin (f), or fibronectin (g); (h-i) The H&E stain images displayed numerous blood vessels and TRAP (Tartrate-Resistant Acid Phosphatase) positive cells; (j) The FITC confocal images revealed blood vessels and fibrous-like tissues in the cKO growth plate; and (k) The entire Bmpr1a -null growth plate was replaced by an ectopic bone-like structure. 
The working hypothesis is that BMPR1A determines the cell fate of the growth plate via SOX9: The progenitor cells proliferate and exit the cell cycle and undergo maturation away from the resting zone to form chondrocytes followed by endochondral bone formation. In the absence of Bmpr1a , these progenitor cells will form either osteoblast cell or fibrous like cells, leading to a mixture structure of bone (dominant) and fibrous tissue in the growth plate. 
Bone morphogenic proteins (BMPs) are critical for both chondrogenesis and osteogenesis. Previous studies reported that embryos deficient in Bmp receptor (Bmpr)1a or Bmpr1b in cartilage display subtle skeletal defects; however, double mutant embryos develop severe skeletal defects, suggesting a functional redundancy that is essential for early chondrogenesis. In this study, we examined the postnatal role of Bmpr1a in cartilage. In the Bmpr1a conditional knockout (cKO, a cross between Bmpr1a flox and aggrecan-CreER (T2) induced by a one-time-tamoxifen injection at birth and harvested at ages of 2, 4, 8 and 20 weeks), there was essentially no long bone growth with little expression of cartilage markers such as SOX9, IHH and glycoproteins. Unexpectedly, the null growth plate was replaced by bone-like tissues, supporting the notions that the progenitor cells in the growth plate, which normally form cartilage, can form other tissues such as bone and fibrous; and that BMPR1A determines the cell fate. A working hypothesis is proposed to explain the vital role of BMPR1A in postnatal chondrogenesis.
 
Administration of anti-4-1BB mAb has been found to be a potent adjuvant when combined with other therapeutic approaches, e.g. chemotherapy, cytokine therapies, anti-OX40 therapy, and peptide or DC vaccines. However, the adjuvant effect of anti-4-1BB mAb administration in adoptive T cell therapy of cancer has not been fully evaluated. In this report, effector T cells were generated in vitro by anti-CD3/anti-CD28 activation of tumor-draining lymph node (TDLN) cells and used in an adoptive immunotherapy model. While T cells or anti-4-1BB alone showed no therapeutic efficacy in mice bearing macroscopic 10-day pulmonary metastases, T cells plus anti-4-1BB mediated significant tumor regression in an anti-4-1BB dose dependent manner. Mice bearing microscopic 3-day lung metastases treated with T cells alone demonstrated tumor regression which was significantly enhanced by anti-4-1BB administration. NK cell depletion abrogated the augmented therapeutic efficacy rendered by anti-4-1BB. Cell transfer between congenic hosts demonstrated that anti-4-1BB administration increased the survival of adoptively transferred TDLN cells. Using STAT4(-/-) mice, we found that modulated IFN gamma secretion in wt TDLN cells after anti-CD3/CD28/4-1BB activation in vitro was lost in similarly stimulated STAT4(-/-) TDLN cells. Additionally, anti-4-1BB administration failed to augment the therapeutic efficacy of T cell therapy in STAT4(-/-) mice. Together, these results indicate that administered anti-4-1BB mAb can serve as an effective adjuvant to augment the antitumor reactivity of adoptively transferred T cells by recruiting the host NK cells; increasing the persistence of infused effector T cells, and modulating the STAT4 molecular signaling pathway.
 
AMPK activation suppresses LXR ligand-mediated induction of Srebp-1c promoter activity in McA-RH7777 cells. A, Diagram of plasmids containing the wild-type or mutant mouse Srebp-1c promoter linked to the firefly luciferase reporter gene. B, Luciferase reporter assay. On Day 0, McA-RH7777 cells were plated at density of 5 x 10 4 cells per well in 24-well plates in Medium A supplemented with 10% FBS and incubated at 37°C in a 5% CO 2 incubator. On Day 1, cells were washed with 0.5 ml PBS and 0.5 ml fresh Medium A was added to each well before transfection. Cells were cotransfected with plasmid D and a control plasmid as described in Materials and Methods. Six hours after transfection, cells were washed with 0.5 ml PBS, switched to Medium A supplemented with 10% delipidated FBS and 0.5 mM AICAR or metformin or various amounts of T0901317, and incubated for 16 hours at 37°C and 5% CO 2 . On Day 2, cells were washed with 0.5 ml PBS, and luciferase and beta-gal activities were measured as described in the Materials and Methods. Normalized firefly luciferase activity without treatment was arbitrarily set as 1.  
Activation of AMPK decreases the fold induction of wild-type and mutant Srebp-1c promoter activities by LXR ligand T0901317 in McA-RH7777 cells treated with compactin. McA-RH7777 cells were cultured, transfected, and treated with AICAR (0.5 mM) or metformin (0.5 mM) with or without T0901317 (1 μM) in the presence of compactin (50 μM compactin + 50 μM sodium mevalonate) as in Fig. 1. Cells were lysed and luciferase reporter assays were performed as in Fig. 1 with (A) wild-type Srebp-1c promoter construct pD, (B) pM31 (-LXRE1, 2), (C) pM24 (-SRE), and (D) pM34 (-LXRE1, 2/SRE). The value on each column represents the fold change (the average of three assays) as compared with control (-AICAR/Metformin/T0901317). White bar, -T0901317; grey bar, + T0901317. *, P<0.05 as compared with control (+ T0901317) without AICAR/metformin treatment for each promoter construct.  
AMPK activation attenuates the fold induction of endogenous SREBP-1c mRNA by LXR ligand T0901317 in McA-RH7777 cells treated with compactin . On Day 0, McA-RH7777 cells were plated at the density of 7 x 10 5 cells per 100-mm dish and cultured in Medium A supplemented with 10% FBS at 37°C in a 5% CO 2 incubator. On Day 2, cells were washed with 1x PBS and cultured for 16 h in Medium A supplemented with 10% delipidated FBS and compactin (50 μM compactin + 50 μM sodium mevalonate) in the absence or presence of additional compounds (AICAR, 0.5 mM; metformin, 0.5 mM; and T0901317, 1 μM). Each treatment was performed in duplicate dishes. On Day 3, cells from duplicate dishes were harvested and combined, total RNA was prepared and reverse transcribed, and endogenous SREBP-1c mRNA levels were measured by real-time PCR as described in Materials and Methods. The value on each column represents the fold change (the average of three assays) as compared with control (-T0901317). White bar, -T0901317; grey bar, + T0901317. *, P<0.05 as compared with control (+ T0901317) without AICAR/metformin treatment.  
AMPK activation attenuates the fold induction of endogenous SREBP-1c mRNA by LXR ligand T0901317 in Fao cells. Fao cells were cultured and treated as McA-RH7777 cells in Fig. 3 except that Medium A was replaced with Medium B and the concentration of both AICAR and metformin was 1 mM. Cells from duplicate dishes were harvested and combined, total RNA was prepared and reverse transcribed, and endogenous SREBP-1c mRNA levels were measured by real-time PCR as described in Materials and Methods. The value on each column represents the fold change (the average of three assays) as compared with control (-T0901317). White bar, -T0901317; grey bar, + T0901317. *, P<0.05 as compared with control (+ T0901317) without AICAR/metformin treatment.  
Activation of AMP-activated protein kinase (AMPK) inhibits hepatic fatty acid synthesis by suppressing sterol regulatory element-binding protein (SREBP)-1c, a master regulator of hepatic lipogenic gene expression. Using a model cell line rat hepatoma McA-RH7777 (CRL-1601) that mimics the behavior of the intact liver by producing high levels of SREPB-1c mRNA and protein, we previously showed that AMPK suppresses hepatic Srebp-1c transcription by inhibiting endogenous liver X receptor (LXR) ligand production and SREBP-1c processing. However, whether AMPK directly inhibits ligand-induced LXR activity remained undetermined. In this study we used a series of mutant Srebp-1c promoter linked to a luciferase reporter to determine the inhibitory mechanism in rat hepatoma McA-RH7777 cells. AMPK activation by either AICAR or metformin decreases Srebp-1c promoter activity by about 75%. Normally, the synthetic LXR ligand T0901317 compound increases the wild-type Srebp-1c promoter activity by about 3-fold, which is similar to that observed in the presence of AICAR or metformin. When endogenous LXR ligand production was blocked by the potent HMG CoA reductase inhibitor compactin, T0901317-induced Srebp-1c promoter activity was decreased by AICAR or metformin treatment. In the mutant Srebp-1c promoter in which two LXR elements are intact but the sterol regulatory element (SRE) is disrupted, the fold inductions of the promoter activity by T0901317 without AMPK activators are significantly higher than those with AMPK activators. Furthermore, AMPK activation attenuates induction of endogenous SREBP-1c mRNA by T0901317. These results indicate that AMPK directly inhibits ligand-induced LXR activity in addition to blocking production of endogenous LXR ligands.
 
Alcoholic liver disease (ALD) has become an important liver disease hazard to public and personal health. Oxidative stress is believed to be responsible for the pathological changes in ALD. Previous studies have showed that insulin, a classic regulator of glucose metabolism, has significant anti-oxidative function and plays an important role in maintaining the redox balance. For addressing the effects and mechanisms of insulin pre-administration on ethanol-induced liver oxidative injury, we investigated histopathology, inflammatory factors, apoptosis, mitochondrial dysfunction, oxidative stress, antioxidant defense system, ethanol metabolic enzymes and lipid disorder in liver of ethanol-exposed mice pretreatment with insulin or not. There are several novel findings in our study. First, we found insulin pre-administration alleviated acute ethanol exposure-induced liver injury and inflammation reflected by the decrease of serum AST and ALT activities, the improvement of pathological alteration and the inhibition of TNF-α and IL-6 expressions. Second, insulin pre-administration could significantly reduce apoptosis and ameliorate mitochondrial dysfunction in liver of mice exposed to ethanol, supporting by decreasing caspases-3 activities and the ratio of Bax/Bcl-2, increasing mitochondrial viability and mitochondrial oxygen consumption, inhibition of the decline of ATP levels and mitochondrial ROS accumulation. Third, insulin pre-administration prevented ethanol-mediated oxidative stress and enhance antioxidant defense system, which is evaluated by the decline of MDA levels and the rise of GSH/GSSG, the up-regulations of antioxidant enzymes CAT, SOD, GR through Nrf-2 dependent pathway. Forth, the modification of ethanol metabolism pathway such as the inhibition of CYP2E1, the activation of ALDH might be involved in the anti-oxidative and protective effects exerted by insulin pre-administration against acute ethanol exposure in mice. Finally, insulin pre-administration deteriorated hepatic steatosis in mice exposed to ethanol might be through SRBEP-1c activation. In summary, these results indicated that insulin pre-administration effectively alleviated liver oxidative injury through anti-inflammatory, anti-oxidative and anti-apoptotic activities but also deteriorated hepatic steatosis through SRBEP-1c activation in mice exposed to ethanol. Our study provided novel insight about the effects and mechanisms of insulin on ethanol-induced liver injury.
 
We previously reported a novel interaction between v-Crk and myosin-1c, and demonstrated that this interaction is essential for cell migration, even in the absence of p130CAS. We here demonstrate a role for Crk-myosin-1c interaction in cell adhesion and spreading. Crk-knockout (Crk (‑/‑)) mouse embryo fibroblasts (MEFs) exhibited significantly decreased cell spreading and reduced Rac1 activity. A stroboscopic analysis of cell dynamics during cell spreading revealed that the cell-spreading deficiency in Crk (‑/‑) MEFs was due to the short protrusion/retraction distances and long persistence times of membrane extensions. The low activity of Rac1 in Crk (‑/‑) MEFs, which led to delayed cell spreading in these cells, is consistent with the observed defects in membrane dynamics. Reintroduction of v-Crk into Crk (‑/‑) MEFs rescued these defects, restoring cell-spreading activity and membrane dynamics to Crk (+/+) MEF levels, and normalizing Rac1 activity. Knockdown of myosin-1c by introduction of small interfering RNA resulted in a delay in cell spreading and reduced Rac1 activity to low levels, suggesting that myosin-1c also plays an essential role in cell adhesion and spreading. In addition, deletion of the v-Crk SH3 domain, which interacts with the myosin-1c tail, led to defects in cell spreading. Overexpression of the GFP-myosin-1c tail domain effectively inhibited the v-Crk-myosin-1c interaction and led to a slight decrease in cell spreading and cell surface area. Collectively, these findings suggest that the v-Crk-myosin-1c interaction, which modulates membrane dynamics by regulating Rac1 activity, is crucial for cell adhesion and spreading.
 
The effects of berberine hydrochloride on the levels of Gck (A), G6pc (B), Pck1 (C) and Srebp-1c (D) transcripts in hepatocytes from SD rat. Primary hepatocytes were treated with either vehicle or berberine hydrochloride (1 to 25 μM) for 6h in the absence or present of 1 nM insulin. The expression levels of indicated transcripts were analyzed using real-time PCR. The expression level in control group without insulin was arbitrarily assigned as 1. Results represented means ± SD of three independent experiments (* for comparing the fold induction of the indicated transcript at the berberine hydrochloride concentration in the absence with that in the presence of insulin; for Gck, b>a; for G6pc, d/e>g, c/d/e>h, d'>f'/g'/h', and e'>g'; for Pck1, n>i/j/k; for Srebp1c, t>s>o/p/q, t>r, and t'>o'/p'/q'/r'/s'; all P<0.05).  
The effects of berberine hydrochloride on the stabilities of 36B4 (A), Gck (B), G6pc (C), Pck1 (D) and Srebp-1c (E) transcripts. ZL rat primary hepatocytes were treated with medium A containing 3 μM α-amanitin in the absence or present of 25 µM berberine hydrochloride. Total RNAs were extracted at 0.5, 1, 2, 4 and 6 h after treatment, and subjected to real-time PCR analysis. The amount of indicated gene transcripts at time 0 was arbitrarily assigned as 100%. Results represented means ± SD of three independent experiments.  
The effects of berberine hydrochloride on the levels of Gck (A), G6pc (B), Pck1 (C) and Srebp-1c (D) transcripts in hepatocytes from Zucker lean rat. Primary hepatocytes from ZL rats were treated with either vehicle or berberine hydrochloride (2.5 to 25 μM) for 6h in the absence or present of 1 nM insulin. Total RNAs were extracted and subjected to real-time PCR analysis. The gene expression level in control group without insulin was arbitrarily assigned as 1. Results represented means ± SD of four independent experiments (* for comparing the fold induction of the indicated transcript at the berberine hydrochloride concentration in the absence with that in the presence of insulin; for Gck, e>c>a, d/e>a/c, a'>c'/d'>e'; for G6pc, f/g/h>i/j, and j'>f'/g'/h'>i'; for Pck1, l>k; for Srebp1c, m>n; all P<0.05).  
The effects of berberine hydrochloride on the levels of Gck (A), G6pc (B), Pck1 (C) and Srebp-1c (D) in hepatocytes from Zucker fatty rat. Primary hepatocytes from ZF rats were treated with either vehicle or berberine hydrochloride (1 to 25 μM) for 6h in the absence or present of 1 nM insulin. Total RNAs were extracted and subjected to real-time PCR analysis. The gene expression level in control group without insulin was arbitrarily assigned as 1. Results represented means ± SD of three independent experiments (* for comparing the fold induction of the indicated transcript at the berberine hydrochloride concentration in the absence with that in the presence of insulin; for Gck, f>d>a, e>a, e/f>b, d'/e'>a'/b'; for G6pc, g>i>k, g>j/l, h>j/k/l, and g'/h'>i'/k'/l'; for Pck1, m>n/r>p/q, and m>o; all P<0.05).  
The effects of berberine hydrochloride on viability of primary hepatocytes from SD rats. The primary hepatocytes from SD rats were first incubated in medium A without or with berberine hydrochloride (1 to 100 μM) for 4 hours in the absence or present of 1 nM insulin. After this pre-incubation, MTT was added for additional 4 hours before hepatocyte viabilities were determined as described in the Materials and Methods section. The OD value in control group without insulin was arbitrarily assigned as 100%. Results represented means ± SD of six experiments (* P < 0.01, for comparing the values of control with the values of the indicated treatments).  
The effects of hormonal and dietary stimuli on hepatic glucose and lipid homeostasis include regulation of gene expression. Berberine, an effective compound in certain Chinese medicinal herbs, has been reported to lower plasma glucose and lipid levels in diabetic and hypercholesterolemic patients. We hypothesized that it may affect the expression of hepatic genes involved in glucose and lipid metabolism. The effects of berberine hydrochloride on viability, gene expression, and activation of AMP activated protein kinase (AMPK) in primary hepatocytes from Sprague-Dawley (SD), Zucker lean (ZL) or fatty (ZF) rats were examined with MTT assay, real-time PCR, and western blotting, respectively. Berberine hydochloride at 50 µM or higher caused cytotoxic effects on hepatocytes. In SD and ZL hepatocytes, it induced Gck and suppressed G6pc expression at 10 and 25 µM, but not as potent as 1 nM insulin. Its effects on Pck1, and insulin-regulated Gck and G6pc expression depended on the hepatocyte sources and the dosage used. In ZF hepatocytes, it increased Gck, and suppressed Pck1 and G6pc expression without insulin. Its effects on Gck and G6pc, but not Pck1 expression, were additive with insulin. Berberine hydrochloride at 25 µM attenuated insulin-suppressed Pck1 (ZL/ZF cells), and insulin-induced Srebp-1c expression (SD/ZL/ZF cells), suggesting modulation of insulin action. Berberine hydrochloride did not alter these genes' mRNA stability. Its treatment caused a dose-dependent increase of phosphorylation of AMPKα, and its substrate, acetyl-CoA carboxylase, in primary hepatocytes. We conclude that berberine hydrochloride regulated the transcription of hepatic genes involved in glucose and fatty acid metabolism.
 
SIRT1, an NAD-dependent histone/protein deacetylase, has classically been thought of as a nuclear protein. In this study, we demonstrate that SIRT1 is mainly localized in the nucleus of normal cells, but is predominantly localized in the cytoplasm of the cancer / transformed cells we tested. We found this predominant cytoplasmic localization of SIRT1 is regulated by elevated mitotic activity and PI3K/IGF-1R signaling in cancer cells. We show that aberrant cytoplasmic localization of SIRT1 is due to increased protein stability and is regulated by PI3K/IGF-1R signaling. In addition, we determined that SIRT1 is required for PI3K-mediated cancer cell growth. Our study represents the first identification that aberrant cytoplasm localization is one of the specific alternations to SIRT1 that occur in cancer cells, and PI3K/IGF-1R signaling plays an important role in the regulation of cytoplasmic SIRT1 stability. Our findings suggest that the over-expressed cytoplasmic SIRT1 in cancer cells may greatly contribute to its cancer-specific function by working downstream of the PI3K/IGF-1R signaling pathway.
 
Schematic Diagram of PBMC Lysate Preparation from Whole Blood for 1 Subject 
Dilutional linearity of PBMC lysates in IP-MSD assay. PBMC lysates were divided into two groups-CSF-1 stimulated (+) and non-stimulated (-). FMS and p-FMS were measured for both the samples using the MSD assay. The samples were sequentially diluted 50% in lysis buffer and each of the diluted samples analyzed in both the FMS and p-FMS assays. The dotted and solid vertical lines display the minimum PBMC lysate volumes (μL) needed for the p-FMS and FMS assays, respectively.
Comparison of p-FMS/FMS between CSF-1 stimulated and unstimulated PBMCs. In order to compare the two assays, fold difference assessed in figure 4 was averaged between the donors (N=5) using the IP-Western and the IP-MSD assay and plotted as shown, with the left y-axis capturing the IP-Western data and the right y-axis capturing the IP-MSD data. 
A novel assay was developed to measure ratio of p-FMS (phospho FMS) to FMS using the Meso Scale Discovery(®) (MSD) technology and compared to the routinely used, IP-Western based approach. The existing IP-Western assay used lysed PBMCs (Peripheral Blood Mononuclear Cells) that were immunoprecipitated (IP) overnight, and assayed qualitatively by Western analysis. This procedure takes three days for completion. The novel IP-MSD method described in this paper employed immunoprecipitation of the samples for one hour, followed by assessment of the samples by a ruthenium labeled secondary antibody on a 96-well Streptavidin-coated MSD plate. This IP-MSD method was semi-quantitative, could be run in less than a day, required one-eighth the volume of sample, and compared well to the IP-Western method. In order to measure p-FMS/FMS, samples from healthy volunteers (HV) were first stimulated with CSF-1(Macrophage colony-stimulating factor) to initiate the changes in the phosphotyrosyl signaling complexes in FMS. The objective of the present work was to develop a high throughput assay that measured p-FMS/FMS semi-quantitatively, with minimal sample requirement, and most importantly compared well to the current IP-Western assay.
 
DOC-1R (deleted in oral cancer-1 related) is a novel putative tumor suppressor. This study investigated DOC-1R antitumor activity and the underlying molecular mechanisms. Cell phenotypes were assessed using flow cytometry, BrdU incorporation and CDK2 kinase assays in DOC-1R overexpressing HeLa cells. In addition, RT-PCR and Western blot assays were used to detect underlying molecular changes in these cells. The interaction between DOC-1R and CDK2 proteins was assayed by GST pull-down and immunoprecipitation-Western blot assays. The data showed that DOC-1R overexpression inhibited G1/S phase transition, DNA replication and suppressed CDK2 activity. Molecularly, DOC-1R inhibited CDK2 expression at the mRNA and protein levels, and there were decreased levels of G1-phase cyclins (cyclin D1 and E) and elevated levels of p21, p27, and p53 proteins. Meanwhile, DOC-1R associated with CDK2 and inhibited CDK2 activation by obstructing its association with cyclin E and A. In conclusion, the antitumor effects of DOC-1R may be mediated by negatively regulating G1 phase progression and G1/S transition through inhibiting CDK2 expression and activation.
 
Measles remains a serious vaccine preventable cause of mortality in developing nations. Vietnam is aiming to achieve the level of immunity required to eliminate measles by maintaining a high coverage of routine first vaccinations in infants, routine second vaccinations at school entry and supplementary local campaigns in high-risk areas. Regular outbreaks of measles are reported, during 2005-2009. National measles case-based surveillance data collected during 2005-June 2009 was analyzed to assess the epidemiological trend and risk factors associated with measles outbreak in Vietnam. Of the 36,282 measles suspected cases reported nationwide, only 7,086 cases were confirmed through laboratory examination. Although cyclical outbreaks occurred between 2005 and 2009, there was no definite trend in measles outbreaks during these periods. Overall, 2438 of measles confirmed cases were among children ≤5 years and 3068 cases were among people ≥16 years. The distribution with respect to gender skewed towards male (3667 cases) significant difference was not observed (P= 0.1693). Unsurprisingly, 4493 of the confirmed cases had no history of vaccination (X(2) <0.01). The northern and highland regions were identified as the main endemic foci and the spatial distribution changed with time. The occurrence of cases, in a considerable proportion of vaccinated population, is not only a reflection of the high vaccination coverage in Vietnam but also portrays a possibility of less than 100% vaccine efficacy. More so, in order to prevent measles in adults, high-risk groups must be identified and catch-up for selected groups selected. This study therefore reinforces the need for continued improvement of surveillance system and to probe into the possible role of changes in age-distribution of cases if the effective control of measles is to be achieved.
 
Dysregulation of certain microRNAs (miRNAs) in cancer can promote tumorigenesis, metastasis and invasion. However, the functions and targets of only a few mammalian miRNAs are known. In particular, the miRNAs that participates in radiation induced carcinogenesis and the miRNAs that target the tumor suppressor gene Big-h3 remain undefined. Here in this study, using a radiation induced thymic lymphoma model in BALB/c mice, we found that the tumor suppressor gene Big-h3 is down-regulated and miR-21 is up-regulated in radiation induced thymic lymphoma tissue samples. We also found inverse correlations between Big-h3 protein and miR-21 expression level among different tissue samples. Furthermore, our data indicated that miR-21 could directly target Big-h3 in a 3'UTR dependent manner. Finally, we found that miR-21 could be induced by TGFβ, and miR-21 has both positive and negative effects in regulating TGFβ signaling. We conclude that miR-21 participates in radiation induced carcinogenesis and it regulates TGFβ signaling.
 
TGF-β1 upregulated miR-21 expression to promote keratinocyte migration in HaCaT cell. (A), miR-21 expression was detected by Northern blot in HaCaT cell treated with or without TGF-β1. (B), MTT assay showed that there was no obvious difference between miR-21-tansfected HaCaT cells (miR-21) and scrambled-miRNA-transfected (Scramble) cells. miR-21 overexpression with mimic were confirmed by Northern Blot. (C), miR-21 promoted cell migration in HaCaT cell. Scratch wound-healing assay was conducted in miR-21-transfected HaCaT cells, and control
MicroRNAs involved in keratinocyte migration and wound healing are largely unknown. Here, we revealed the indispensable role of miR-21 in keratinocyte migration and in re-epithelialization during wound healing in mice. In HaCaT cell, miR-21 could be upregulated by TGF-β1. Similar to the effect of TGF-β1, miR-21 overexpression promoted keratinocyte migration. Conversely, miR-21 knockdown attenuated TGF-β1-induced keratinocyte migration, suggesting that miR-21 was essential for TGF-β-driven keratinocyte migration. Furthermore, we found that miR-21 was upregulated during wound healing, coincident with the temporal expression pattern of TGF-β1. Consistently, knockdown of endogenous miR-21 using a specific antagomir dramatically delayed re-epithelialization possibly due to the reduced keratinocyte migration. TIMP3 and TIAM1, direct targets of miR-21, were verified to be regulated by miR-21 in vitro and in vivo, indicating that these two molecules might contribute to miR-21-induced keratinocyte migration. Taken together, our results demonstrate that miR-21 promotes keratinocyte migration and boosts re-epithelialization during skin wound healing.
 
Interleukin-6 (IL-6) levels are known to be increased in patients with rheumatoid arthritis (RA). Tocilizumab, a monoclonal antibody to the IL-6 receptor (IL-6R), reduces disease activity in RA, although its mechanisms of action remain unclear. Since IL-6 regulates cytokine production by CD4 T cells during activation, we investigated whether treatment with tocilizumab altered the phenotype and cytokine production by CD4 T cells in patients with rheumatoid arthritis. We show here that tocilizumab treatment does not change the production of cytokines by naïve CD4 T cells. However, tocilizumab treatment causes a selective decrease of IL-21 production by memory/activated CD4 T cells. Since IL-21 is known to promote plasma cell differentiation, we examined the effect of tocilizumab on the production of autoantibodies. We show that there is a decrease in the levels of IgG4 anti-CCP antibodies, but there is no effect on IgG1 anti-CCP antibodies. In addition, we show that IL-21 is a powerful inducer of IgG4 production by B cells. Thus, IL-6 contributes to the presence of IgG4-specific anti-CCP autoantibodies in RA patients, likely through its effect on IL-21 production by CD4 T cells, and IL-6R blockade down-regulates this pathway.
 
Norcantharidin (NCTD), a demethylated form of cantharidin, has been used as a routine anticancer drug in China. In this study, the effect and mechanism of NCTD on anti-hepatocellular carcinoma (HCC) was examined. In vivo antitumor activity was investigated in hepatoma-bearing mice by intraperitoneal injection of different concentration of NCTD. The levels of MicroRNAs (miRNAs) and mRNA were detected by real-time PCR. The concentrations of IL-10 and IL-12 in BMDMs, Raw 264.7 cells or tumor-associated macrophages (TAMs) were measured with ELISA kit. The effects of TAMs on H22 cell survival and invasion were assayed via the CCK-8 and tumor invasion assay, respectively. Anti-miR-214 or pre-miR-214 was used to down-regulate or up-regulated miR-214 expression. The results showed that NCTD drastically impaired tumor growth in hepatoma-bearing mice, correlating with increased anti-tumor activity of TAMs. Moreover, NCTD stimulation led to an alteration of HCC microenvironment, reflected by a decrease in a shift from M2 to M1 polarization and the populations of CD4+/CD25+Foxp3 T cells. The activation of STAT3 was inhibited in TAMs from hepatoma-bearing mice injected with NCTD. Addition of NCTD to treat RAW264.7 or TAMs enhanced M1 polarization through increase of miR-214 expression. NCTD significantly inhibited β-catenin expression, which could be reversed by miR-214 inhibitor. Conditioned media from TAMs in hepatoma-bearing mice treated with NCTD or TAMs transfected with pre-miR-214 inhibited survival and invasion of H22 cells. This finding reveals a novel role for NCTD on inhibition of HCC through miR-214 modulating macrophage polarization.
 
Pancreatitis occurs when digestive enzymes are activated in the pancreas. Severe pancreatitis has a 10-30% mortality rate. No specific treatments for pancreatitis exist now. Here, we discovered that interleukin-22 (IL-22) may have therapeutic potential in treating acute and chronic pancreatitis. Wild-type and IL-22 knockout mice were equally susceptible to cerulein-induced acute and chronic pancreatitis, whereas liver-specific IL-22 transgenic mice were completely resistant to cerulein-induced elevation of serum digestive enzymes, pancreatic necrosis and apoptosis, and inflammatory cell infiltration. Treatment of wild-type mice with recombinant IL-22 or adenovirus IL-22 markedly attenuated the severity of cerulein-induced acute and chronic pancreatitis. Mechanistically, we show that the protective effect of IL-22 on pancreatitis was mediated via the induction of Bcl-2 and Bcl-X(L), which bind to Beclin-1 and subsequently inhibit autophagosome formation to ameliorate pancreatitis. In conclusion, IL-22 ameliorates cerulein-induced pancreatitis by inhibiting the autophagic pathway. IL-22 could be a promising therapeutic drug to treat pancreatitis.
 
Hypovitaminosis D is highly prevalent in chronic kidney disease (CKD). Recently, vitamin D has sparked widespread interest because of its potential favorable benefits on cardiovascular disease (CVD). Evidence from clinical studies and animal models supports the existence of biphasic cardiovascular effects of vitamin D, in which lower doses suppress CVD and higher doses stimulate CVD. However, the mechanism for the different effects remains unclear. Fibroblast growth factor-23 (FGF-23) is a recently identified member of the FGF family, and thought to be actively involved in renal phosphate and vitamin D homeostasis. More specifically, Vitamin D stimulates FGF-23 secretion and is inhibited by increased FGF-23. Given this background, we hypothesize that FGF-23 may provide a unique tool to explain the biphasic cardiovascular effects of vitamin D in CKD. The data presented in this review support the hypothesis that FGF-23 may be linked with the high cardiovascular risk in CKD through accelerating the onset of vascular calcification, secondary hyperparathyroidism, left ventricular hypertrophy and endothelial dysfunction. Therefore, modulation of FGF-23 may become a potential therapeutic target to lowing cardiovascular risk in CKD. Several clinical interventions, including decreased phosphate intake, phosphate binders, cinacalcet plus concurrent low-dose vitamin D, C-terminal tail of FGF-23 and renal transplantation, have been employed to manipulate FGF-23.
 
This study examined the effects of parathyroid hormone-related protein (PTHrP) derived from human MDA-MB-231 breast cancer cells on the tumor growth and osteoblast inhibition. Results revealed that knocking down PTHrP expression in the breast cancer cells strikingly inhibited the formation of subcutaneous tumors in nude mice. PTHrP knockdown dramatically decreased the levels of cyclins D1 and A1 proteins and arrested the cell cycle progression at the G1 stage. PTHrP knockdown led to the cleavage of Caspase 8 and induced apoptosis of the tumor cells. Interestingly, knocking down PTHrP increased the levels of Beclin1 and LC3-II and promoted the formation of autophagosomes. Knocking down PTHrP expression significantly reduced the abilities of the breast cancer cells to inhibit osteoblast differentiation and bone formation in vitro and in vivo. Finally, we found that PTHrP activated its own expression through an autocrine mechanism in MDA-MB-231 cells. Collectively, these studies suggest that targeting PTHrP expression in the tumor cells could be a potential therapeutic strategy for breast cancers, especially those with skeletal metastases.
 
Flow cytometry analysis of 24p3 protein on the L929 cell cycle after 96 h incubation. 
Detection of 24p3 protein induced L929 cells DNA damage. Cells were cultured in cover slips with 10 μM 24p3 protein supplement for 96h; the formation of DNA damage can be observed by cell cycle measurement. Upper panel, Control; Lower panel, 10 μM 24p3 protein treated cells. After treatment with 24p3 protein, cells were detached by trypsinization and stained with PI then determined cell cycle by flow cytometry.
Flow cytometry analysis of 24p3 protein on the L929 cell cycle after 96 h incubation.
It is well known that mouse uterine 24p3 protein, is an acute phase protein, secreted from the L929 cell line, and that it will be induced by the dexamethasone stimulation of the cell. We investigated the possible effects of 24p3 protein on the L929 cell line, by observing its morphological change, ROS increase and viability decrease, by the process of culturing in a 24p3 protein-supplemented medium. Following the L929 cells' exposure to the 24p3 protein supplement for a period of 72 hours, S-phase cells accumulated to a significant degree, suggesting that the entry into the G2/M phase from the S phase, in the cell cycle progression, was blocked. There was a significant decrease in cell numbers and increased DNA damage within the cells in the presence of 24p3 protein within the medium for 96 hours, implying that they have undergone pathway of cell death. After 96h incubation in low concentration of 24p3 protein, the result of PI/annexin V double staining showed cell death obviously. These results suggest that 24p3 protein-induced S phase arrest in the cell cycle, would cause DNA damage, followed by cell death in the L929 cells.
 
Clinical Characteristics of Patients in Three Independent Cohorts.
The stability and reproducibility of multiplex-RT qPCR. (A, B) Pearson correlation of U6B-normalized miR-26a (A) and miR-26b (B) readings from multiplex- and singleplex-RT qPCR in cohort 1 (n=129). (C, D) Pearson correlation of six individual reference-normalized miR-26 readings derived from SBC and NCI. The multiplex-RT qPCR assay was performed in the same RNA isolates (n=10) (C) or different RNA isolates from two different tumor regions of the same FFPE specimens (n=10) (D). (E, F) Forest plots showing hazard ratios in log2 scale for the overall survival of HCC patients from cohort 1 treated with IFNα versus controls without or with selection by U6B-normalized miR-26a (E) or miR-26b (F). The median of U6B-normalized miR-26a and miR-26b were used as cut-off points.
Development of a template MIR26-DX to identify candidate HCC patients for adjuvant IFNα therapy based Cohort 1. (A) A schematic diagram of the MIR26-DX. A 6-point scoring system was developed based on the median miR-26 expression normalized to six reference transcripts in the Cohort 1 as described in the Method section. (B) The forest plot showing hazard ratios in log2 scale for the overall survival of HCC patients from Cohort 1 treated with IFNα versus controls without or with selection by the 6-point score system. (C) Kaplan-Meier survival curves showing the overall survival of HCC patients with low miR-26 (left) or high miR-26 (right) from Cohort 1 treated with or without IFNα. The number of cases under risk was listed.
Association between miR-26 expression in tumors and overall survival of HCC patients treated with IFNα. Data were shown in Cohort 2 (panels A-C) or in Cohort 3 (panels D-F). The case assignment of high or low miR-26 was based on the 6-point score system. No assignment (A, D); cases assigned as low miR-26 (B, E); cases assigned as high miR-26 (C, F). The number of cases under risk was listed.
The Prognostic ROC curve analysis for the survival of HCC patients in response to adjuvant IFNα therapy.
Background & Aims: Adjuvant therapies for hepatocellular carcinoma (HCC) such as interferon-alpha are effective only in a subset of patients. Previously we found that HCC patients with low level of miR-26 have survival benefits from interferon-alpha. The purpose of this study is to develop a standardized miR-26 diagnostic test (referred as MIR26-DX) to assist identification of candidate HCC patients for adjuvant interferon-alpha therapy. Methods: We developed a multiplex reverse-transcription quantitative polymerase-chain-reaction assay to determine the levels of two HCC-related miR-26 transcripts along with six small RNA reference transcripts. We evaluated archived paraffin-embedded tissues from three cohorts of HCC patients (n=248) who underwent radical resection at three different clinical centers. Fifty-two percent of them underwent adjuvant interferon-alpha therapy. We used Cox-Mantel log-rank test to evaluate patient survival. Results: We found that the multiplexing assay was stable and reproducible regardless of differences in sample preparations and operators. We developed a matrix template and a scoring algorithm based on a training cohort (n=129) to assign HCC patients, and then applied the template in two test cohorts (n=119). The proportions of HCC patients assigned as low miR-26 by this algorithm were 68, 4, and 63 percent in the training cohort and two test cohorts, respectively. Consistently, HCC with low miR-26 had a favorable response to interferon-alpha with improved median overall survival (≥3year). Conclusions: MIR26-DX is a simple and reliable companion diagnostic test to select HCC patients for adjuvant interferon-alpha therapy.
 
Span 80 (sorbitan monooleate) vesicles behaved differently from conventional phospholipid vesicles (liposomes) because the former had a more fluid interface. After doxorubicin hydrochloride (DOX) was encapsulated into the Span 80 vesicle (loading efficiency: 63 %), DOX-loaded Span 80 vesicles (DVs) were thereafter added to Colon 26 cells. It was suggested, from the flow cytometric analysis and confocal laser microscopic observation, that DVs directly deliver DOX into the cytoplasm of Colon 26 cells. DVs showed the different delivery manner from the DOX-loaded liposomes (DLs). It is considered that the difference of delivery manner between DVs and DLs resulted in the difference of cytotoxicity (IC(50)); i.e. IC(50) values for DVs and DLs were 5 and > 30 μM, respectively. The results obtained herein would give the fundamental findings which can contribute to the improvement of formulation of conventional liposome-based carrier and its cytotoxicity.
 
Signaling involved in IL-27 expression. IL-27 is largely produced by Antigen-presenting cells (APCs) such as Dendritic cells (DCs) and macrophages upon stimulation with TLRs agonists, IFN- α , IFN- γ or microbial infections. It consists of two subunits (p28/EBI3) which are expressed independently. TLR2, TLR4 and TLR9-associated MyD88 can induce EBI3 expression through the binding of NF- κκ B subunits (p50/p65) and PU.1 to the EBI3 promoter. TLR4-associated MyD88 induces p28 expression through binding of NF- κκ B-c-Rel and AP-1/c-Fos to the p28 promoter, TLR4-associated TRIF induces p28 expression by binding of IRF3 to the p28 promoter. The IFN- α and IFN- γ induces p28 expression through the binding of IRF3 and IRF8 to the p28 promoter, respectively. In addition, IFN- γ -mediated IL-27 instead of IL-27p28 gene expression is positively regulated by the C-Jun N-terminal kinases (JNK), mitogen-activated protein kinases (MAPKs) and the phosphoinositide 3-kinase (PI3K). 
IL-27, a heterodimeric cytokine of IL-12 family, regulates both innate and adaptive immunity largely via Jak-Stat signaling. IL-27 can induce IFN-γ and inflammatory mediators from T lymphocytes and innate immune cells. IL-27 has unique anti-inflammatory properties via both Tr1 cells dependent and independent mechanisms. Here the role and biology of IL-27 in innate and adaptive immunity are summarized, with special interest with immunity against Mycobacterium tuberculosis.
 
Functional loss of both alleles of the breast cancer susceptibility gene, BRCA2, facilitates tumorigenesis. However, the direct effects of BRCA2 heterozygosity remain unclear. Here, BRCA2 heterozygosity was mimicked in HT-29 colon cells by reducing levels of BRCA2 through stable RNA interference. No difference in RAD51 subcellular localization and focus formation was observed between control and mimicked heterozygous cell lines. DNA repair ability, as measured by colony survival following mitomycin C treatment and ultraviolet radiation exposure, was also unaffected by reduced levels of BRCA2. Interestingly, the growth rate of the mimicked BRCA2 heterozygous cell line was significantly lower than that of control cells. Increased expression of p53 in the mimicked heterozygous cells was observed, perhaps in response to BRCA2 deficiency. Levels of p27 were also found to be slightly increased in cells with reduced BRCA2, perhaps contributing to the slower growth rate. Overall, these results suggest that tumors are unlikely to arise directly from BRCA2 heterozygous cells without other genetic events such as loss of the wild-type BRCA2 allele and/or loss of p53 function or other cell cycle inhibitors.
 
The amiloride-sensitive epithelial sodium channel (ENaC), a plasma membrane protein mediates sodium reabsorption in epithelial tissues, including the distal nephron and colon. Syntaxin1A, a trafficking protein of the t-SNARE family has been reported to inhibit ENaC in the Xenopus oocyte expression and artificial lipid bilayer systems. The present report describes the regulation of the epithelial sodium channel by syntaxin1A in a human cell line that is physiologically relevant as it expresses both components and also responds to aldosterone stimulation. In order to evaluate the physiological significance of syntaxin1A interaction with natively expressed ENaC, we over-expressed HT-29 with syntaxin1A constructs comprising various motifs. Unexpectedly, we observed the augmentation of amiloride-sensitive currents with wild-type syntaxin1A full-length construct (1-288) in this cell line. Both gammaENaC and neutralizing syntaxin1A antibodies blocked native expression as amiloride-sensitive sodium currents were inhibited while munc18-1 antibody reversed this effect. The coiled-coiled domain H3 (194-266) of syntaxin1A inhibited, however the inclusion of the transmembrane domain to this motif (194-288) augmented amiloride sensitive currents. More so, data suggest that ENaC interacts with multiple syntaxin1A domains, which differentially regulate channel function. This functional modulation is the consequence of the physical enhancement of ENaC at the cell surface in cells over-expressed with syntaxin(s). Our data further suggest that syntaxin1A up-regulates ENaC function by multiple mechanisms that include PKA, PLC, PI3 and MAP Kinase (p42/44) signaling systems. We propose that syntaxin1A possesses distinct inhibitory and stimulatory domains that interact with ENaC subunits, which critically determines the overall ENaC functionality/regulation under distinct physiological conditions.
 
The activation of extracellular signal-regulated kinases (ERK1/2) has been associated with specific outcomes. Sustained activation of ERK1/2 by nerve growth factor (NGF) is associated with translocation of ERKs to the nucleus of PC12 cells and precedes their differentiation into sympathetic-like neurons whereas transient activation by epidermal growth factor (EGF) leads to cell proliferation. It was demonstrated that different growth factors initiating the same cellular signaling pathways may lead to the different cell destiny, either to proliferation or to the inhibition of mitogenesis and apoptosis. Thus, further investigation on kinetic differences in activation of certain signal cascades in different cell types by biologically different agents are necessary for understanding the mechanisms as to how cells make a choice between proliferation and differentiation. It was reported that chitinase 3-like 1 (CHI3L1) protein promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts similarly to insulin-like growth factor 1 (IGF1). Both are involved in mediating the mitogenic response through the signal-regulated kinases ERK1/2. In addition, CHI3L1 which is highly expressed in different tumors including glioblastomas possesses oncogenic properties. As we found earlier, chitinase 3-like 2 (CHI3L2) most closely related to human CHI3L1 also showed increased expression in glial tumors at both the RNA and protein levels and stimulated the activation of the MAPK pathway through phosphorylation of ERK1/2 in 293 and U87 MG cells. The work described here demonstrates the influence of CHI3L2 and CHI3L1 on the duration of MAPK cellular signaling and phosphorylated ERK1/2 translocation to the nucleus. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 that leads to a proliferative signal (similar to the EGF effect in PC12 cells), activation of ERK1/2 phosphorylation by CHI3L2 (similar to NGF) inhibits cell mitogenesis and proliferation.
 
Top-cited authors
Yu-Tao Xiang
  • University of Macau
Wensheng Qin
  • Lakehead University Thunder Bay Campus
Chu-Xia Deng
  • Washington University in St. Louis
Teris Cheung
  • The Hong Kong Polytechnic University
Yi-Ping Li
  • University of Alabama at Birmingham