The Research In The Role Of MiR17 Gene In BMP9-Induced Osteogenic Of MSCs

Objective Bone defect is a common disease in clinical study,mainly caused by congenital disease,trauma and postoperative infection.The treatment methods of bone defect mainly include autologous bone graft?bone allograft and bone tissue engineering.As autologous bone grafts and allogeneic bone grafts have the risk of difficultly healing and easy rejection,therefore the bone tissue engineering as a new technology which is being applied to the treatment of bone defect.BMP9 as a powerful osteogenic induction factor which can promote the differentiation of bone marrow mesenchymal stem cells into osteoblasts.Therefore it plays a key role in the treatment of bone defect.In the early stage,we screened related miRNA in the bone formation process of mesenchymal stem cells induced by BMP9 through microRNA chip technology,and found that multiple members of miR-17-92 gene cluster,including miR-17,were down-regulated.Through in vivo and in vitro osteoblastic experiments,there was proved that miR-17 can effectively inhibit BMP9-induced osteoblastic differentiation of MSCs.The text mainly discussed the role of miR-17-5p(or miR-17),in the osteogenesis of BMP9-induced mesenchymal stem cells iMEF.Hoping to provide a certain molecular biological basis for the clinical diagnosis of bone defect through our study.Methods pMPB-OMIR as the control group plasmid were constructed based on Piggybac system.The experimental groups were named miR17-OMIR and Sponge17-OMIR;After the stable transfection of the recombinant plasmid and the empty plasmid with the target gene into the iMEF cells line,the green fluorescent protein was highly expressed,suggesting that the stable cell line was successfully constructed;The target protein genes of miR-17 were predicted by bioinformatics method,and three potential downstream target genes were selected.The target downstream target genes were determined by dualluciferase reporter gene experiment;In order to further verify the regulatory effect of miR-17 gene on downstream target genes,the RB1 mutant was constructed and dual-luciferase experiment was conducted again;ALP staining was used to verify early osteogenesis and alizarin red staining to verify late calcium nodule formation;Bone masses were formed in nude mice by subcutaneous injection,and bone tissue masses were stained with Masson tricolor and HE staining in order to observe and detect the number of osteoblasts and bone maturity in the masses.Results(1)The osteoblastic experiments in vivo and in vitro confirmed that miR-17 gene can inhibit BMP9-induced osteoblastic differentiation of MSCs,and Sponge-17 can partially restore its inhibitory effect.(2)Sequencing verified the construction of recombinant plasmid RB1 3'UTR-pMIR?BMPR2 3'UTR-pMIR?SMAD7 3'UTR-pMIR successfully.(3)Dual-luciferase reporter gene assay showed that RB1 was a direct downstream target gene of miR-17,while BMPR2 and SMAD7 were not direct downstream target genes of miR-17.Cell relative luciferase activity of RB1-3 'UTR(1.137±0.1073)was significantly lower than that of control group pMPB-OMIR(1.517±0.01314)(P < 0.001).(3)The osteogenic experiments in vivo and in vitro further demonstrated that RB1 can partially reverse the osteogenic effect of miR-17 in BMP9 induced MSCs.Conclusion miR-17 gene has a certain inhibitory effect in BMP9-induced bone formation of mesenchymal stem cells,which may be achieved through its downstream direct target gene RB1.Research significance We have found that miR-17 can inhibit the mechanism by which BMP9-induced bone formation of mesenchymal stem cells in vivo and in vitro.Next,we will determine enhance the effect of BMP9 on bone formation of mesenchymal stem cells through targeted regulation of miR-17.We hoped that this research will provide a solid experimental basis for the development of more efficient growth factors.