The Effects And Mechanisms Of MiR-155 On Osteogenetic Differentiation Of Mesenchymal Stem Cells Induced By BMP9

Objective: To investigate the expression of miR-155 during the osteogenic differentiation induced by BMP9,the effect of miR-155 on BMP9 inducing mesenchymal stem cells(MSCs)osteogenesis,and explore the related mechanisms containing in this process.Methods: Used adenovirus BMP9(BMP9)to infect C2C12 cells and MEF cells to induced osteogenic differentiation,tested the expression of miR-155 by quantitative real-time PCR(q RT-PCR)at 0d,1d,3d,5d,7d of differentiation,and detected the mRNA expression levels of Runx2 and ALP simultaneously.Used miR-155 mimic to overexpress or miR-155 inhibitor to inhibit the expression of miR-155,detected the effect of it on the early stage of differentiation by ALP staining and ALP activity,and the effect of it on the later stage of differentiation by Alizarin Red S(ARS)staining.Tested osteogenesis-related genes by q RT-PCR,include Runx2,OSX,ALP and OCN to estimate the effect of miR-155 on mRNA levels.Detected the expression of Runx2,p-Smad1/5/8 and the later osteogenesis makers OCN and OPN on protein levels used Western blot.Used bioinformatics methods to predict the potential target genes of miR-155,selected Runx2 and BMPR2 for deeply analyze,which are closely related the osteoinductive effect of BMP9.qRT-PCR and Western blot were used to validate the effect of miR-155 on the expression of its target genes on mRNA and protein levels respectively.Confirmed the target genes of miR-155 by luciferase reporter assay.Used qRT-PCR to test the mRNA levels of bone-related genes after interfering the target genes of miR-155,studied the role of them in this process.microCT detected ectopic bones collected from subcutaneous ectopic osteogenesis of MSCs in nude mice,made slices for HE staining and Masson staining.Results: During the process of osteogenic differentiation of C2C12 cells and MEF cells induced by BMP9,the expression of miR-155 exhibited a tendency of increasing at first and then decreasing.Overexpressed miR-155 decreased ALP staining,ALP activity and ARS staining.The results of qRT-PCR shown tha miR-155 can repress the expression of bone-related genes,such as Runx2,OSX,ALP and OCN,inhibited the expression of miR-155 could reverse its inhibitory effect.The results of Western blot shown that miR-155 significantly inhibited the expression of Runx2 and p-Smad1/5/8,markedly decreased the expression of OCN and OPN,the later stage of osteogenic differentiation makers,also.miR-155 had no significant influences of its predicted target genes Runx2 and BMPR2 on the mRNA levels from the results of qRT-PCR,while,it could obviously decrease the expression of Runx2 and BMPR2 on the protein levels from the results of Western blot.Luciferase reporter assay shown that miR-155 can directly target Runx2 and BMPR2.qRT-PCR tested the osteogenesis-related genes after interfering Runx2 and BMPR2 respectively,shown interfered the target genes of miR-155 decreased the expression of osteogenesis-related genes dramatically,which had the same function as overexpression of miR-155.The results of microCT revealed that miR-155 could suppress the volume and density of the heterotopic bones derived from MEF cells differentiation induced by BMP9 in vivo,HE staining and Masson staining shown miR-155 inhibited the maturity of ectopic bones.Conclusions: miR-155 could inhibit the osteogenic differentiation induced by BMP9 in mesenchymal stem cells,it may through Smad/BMP signaling pathway to play the inhibitory effect in this process.