| Part One Effect of MicroRNA-34c/PI3K-Akt loop on the regulation of osteogenic differentiation of MC3T3-E1downstream of VaspinBackgroundOsteoporosis (OP) is a common disease which mainly chara-cterized by decreased bone mass and disorder of the bone microstructure. According to epidemiology investigations, the incidence of OP in China is about13.2%, and the costs of OP treatment and prevention brought huge burden to the whole country and society. Adipokines, including adiponectin, leptin, omentin and so on, is a series of activity cytokines secreted by adipose cells. They play an important role in the regulation of bone metabolism. Vaspin is a newly discovered adipokine, and the previous research found that Vaspin could inhibit the apoptosis of osteoblasts. However, the role of Vaspin during the osteogenic differentiation process is still unclear.ObjectiveIn this study, we aimed to investigate the effect of Vaspin on the osteogenic differentiation of MC3T3-E1and find out whether miRNAs were involved in the procedure. Furthermore, we studied the mechanism of how Vaspin affected the miRNA expression for a better understanding of relationship between epigenetic and osteoporosis.MethodsMC3T3-E1cells were treated with different concentration of Vaspin, and the total RNA and protein were isolated for detecting the osteogenic markers using p-NP methods, radioimmunoassay, and western blot. Meanwhile the difference of miRNAs expression in the two groups was tested by microarray technology. Selecte an osteogenic related miRNA and confirme the result with qRT-PCR. Use miRNA mimics or inhibitor transfection to build an over-expression or low-expression model. Detect the osteogenic marker in the models to confirm the effect of miRNA on the osteogenic differentiation. Predict the target gene by software and published articles. Investigate the expression of target gene mRNA and protein in different models to confirm the target gene. Ues western blot to detect the expression of p-Akt in different time point. Using PI3K-Akt pathway inhibitor LY294002to block the pathway and detect the expression of miRNA by qRT-PCR after the blockage. Detecte the expression of osteogenic marker of the LY294002pretreated MC3T3-E1after Vaspin administration. Detect the expression of p-Akt and c-met protein as well as the c-met mRNA in the miR-34c low-expression model.Results1. The ALP activity, OC secretion and Runx2protein expression were decreased in a dose-depended way after72hours incubation of Vaspin. And after20days treatment of Vaspin, the number of mineral node in the β-GP induced MC3T3-E1was less than the control group;2. The expression of mmu-miR-127, mmu-miR-34c, mmu-miR-214, mmu-miR-139, mmu-miR-6350, mmu-miR-30a, mmu-miR-146b, and mmu-miR-483was decreased while mmu-miR-210, mmu-miR-31, mmu-miR-6380, mmu-miR-126, mmu-miR-218-1, mmu-miR-720, mmu-miR-379, and mmu-miR-2861was increased according to the microarray result. Combining our result with the published articles, we chose the miR-34c as the item in the following study. The qRT-PCR result confirmed that the expression of miR-34c was elevated in MC3T3-E1after Vaspin administration;3. We successfully built the low-expression model by transfection of miR-34c inhibitor and we found that the osteogenic inhibiting effect of Vaspin was diminished in this model;4. The Runx2protein expression was increased in the low-expression model while decreased in the over-expression model. However, the mRNA expression was no significant difference in all groups;5. Vaspin could stimulate the expression of p-Akt in MC3T3-E1. Pretreatment with PI3K-Akt inhibitor LY294002could abolish the PI3K-Akt signal pathway as well as decline the expression of miR-34c caused by Vaspin;6. The osteogenic inhibiting effect of Vaspin was diminished after the pretreatment with LY294002;7. The expression of p-Akt as well as the c-met protein were increased in the miR-34c low-expression model, however, the c-met mRNA had no change;Conclusion1. Vaspin could inhibit the osteogenic differentiation of MC3T3-E1in a dose-depended way;2. The increase of miR-34c was a possible mechanism involved;3. The miR-34c and PI3K-Akt could interacte with each other and constitute a regulation loop involved the modulation of MC3T3-E1osteogenic differentiation downstream of Vaspin; Part Two Effects of miR-34c on the spontaneous osteogenic differentiation of human mesenchymal stem cells and the mechanisms involvedBackgroundBone marrow mesenchymal stem cell (MSC) is a series of self-renewal and polypotential cells which can differentiate into osteoblast, adipoblast and chondroblast. As the main source of osteoblasts in human body, MSC lays a great importance on the maintenance of the osteoblast number. Recently studies found that with aging, the number of MSC was gradually declined. Meanwhile, studies also found that spontaneously osteogenic differentiation would happen in MSC during the aging. However, the mechanism related was still unclear. MiR-34c is a newly discovered miRNA, it plays an important role during the stem cell differentiation and bone metabolic regulation. In that case, we speculated that the changing of miR-34c expression may be one of the possible mechanisms in the spontaneous osteogenic differentiation of MSC.ObjectiveIn our study, we investigated the chang of the miR-34c expression and the methylation status alternation of the miR-34c promoter, trying to explain the mechanism involved in the spontaneous osteogenic differentiation of human mesenchymal stem cell.MethodsWe use natural passage for setting of the aging MSC model; Use the p-NP methods, radioimmunoassay, and western blot for detecting the osteogenic markers; Use qRT-PCR to examine the miR-34c level; Use miR-34c mimics or inhibitor transfection for over-expression model or low-expression model and detect the osteogenic markers for analysis of the osteogenic capability in the models; Use Bisulfite Sequencing PCR(BSP) technology to test the methylation level of the miR-34c promoter. Use qRT-PCR for testing DNMTs expression level; Use5-Aza-dC incubating the aging MSC for3days and6days, exact the total RNA for detecting miR-34c, DNMTs mRNA expression.Results1. We successfully isolated MSC and constructed a in vitro amplification model;2. We identified the MSC by flow cytometry as well as dyeing of Alizarin red S and Oil red O after induction;3. With MSC passaging, the ALP activity, OC secretion and Runx2protein expression were increased;4. With MSC passaging, miR-34c expression was decreased. MiR-34c mimics transfection built an over-expression model. In this model, miR-34c expression was increased while osteogenic markers were decreased;5. BSP result showed that methylation of miR-34c promoter was significantly increased during the passaging.6. With MSC passaging, the expression of DNMT1was increased while the DNMT3A and DNMT3B were the same.7. After incubating with5-Aza-dC, the expression of miR-34c was increased while DNMTlwas decreased.ConclusionThe decline of miR-34c caused by methylation is one of the possible mechanisms for the spontaneous osteogenic differentiation of MSC. |
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