Biological Funcations And Molecular Mechanism Of MiR-24in Osteogenic Differentiation Of Human Mesenchymal Stem Cells

Objective:miRNA (microRNA) are very short length of non-coding RNA, by reducing the stability of target genes or inhibit its translation level to influence cell differentiation, proliferation, and apoptosis. MiRNAs have been shown to offer great potential in the ossification process. We seek to investigate the role of miR-24in osteogenic differentiation of human stromal (mesenchymal) stem cells in vitro and elucidate the mechanism of miR-24promating osteogenic differentiation.Methods:The human BMSCs were collected from bone marrow and incubated in vitro for primary culture and passage culture. Detect the purified MSCs with sixthed generation by Flow cytomety. Osteogenic differentiation was induced by the addition of300ng/ml BMP-2. Expression of miR-24in hMSCs cells was detected before and after treatment with BMP2in different time by Northern blotting. miR-24、 anti-miR-24and miR-NC were separately transfected into the BMP-2treatment and nontreated hMSCs by using cationic liposomes. ALP activity was assayed by a spectrophoto-metricmethod using a Lab Assay TMALP kit. Mineration of hMSCs was observed by Alizarin red staining. The protein of Tcf-1and Runx2was detected through western blot. The expression of Tcf-1mRNA was detected by RT-PCR. To create wild type (WT)、mutate(MUT)、deleated(DEL) Tcf-1mRNA3’UTR luciferase reporter vector, These three reporter vectors were cotransfected with miR-24or miR-NC to hMSCs cells, and the luminescent signal was quantified by Dual Luciferase Reporter Assay System.Result:The bone marrow mesenchymal stem cells gradually purified after isolated and cultured primary hMSCs were passaged for many generations, in the early stage of growth the cells formed colonies, the cells were uniform shape and distribution after the sixth generation. And flow cytometry showed that cell populations had homogeneous volume, These results show that the isolated and cultured cells had the same antigenic characteristics of hMSCs. Expression of miR-24decreased progressively after treatment with BMP-2. ALP activity and mineralization determined by Alizarin red staining in cells transfected with miR-24were suppressed. The transfection of miR-24decreases the proteins of Tcf-1and Runx2. The levels of Tcf-1mRNA examined by real time PCR remained nearly unchanged with the treatment of miR-24. Compared to miR-24, The levels of luciferase activity in cells transfected with miR-24and WT-pGL3-Tcf-1mRNA3’UTR were significantly reduced, whereas the activity level of MUT and DET were not reduced.Conclusion:The morphology isolated and cultured primary hMSCs were normal, the hMSCs had the same antigenic characteristics.2. The expression of miR-24decreased progressively during the osteogenic differentiation of hMSCs with the treatment of BMP-2.3. With BMP-2treatment, transfection of miR-24decreases the protein expression levels of Tcf-1and Runx2and Tcf-1mRNA dose’nt changed. miR-24modulate the expression of Tcf-1at a post-translational level. ALP activity and mineralization determined by Alizarin red staining in cells transfected with miR-24were suppressed.4. We have succeed constructed WT、MUT and DEL Tcf-1mRNA3’-UTR Luciferase reporter vector. Tcf-1is a target gene of miR-24by using luciferase reporter assay.