The Role And Mechanism Of MiR-92b-3p In Osteogenic Differentiation Of MC3T3-E1 Cells Induced By BMP-2

Background and purposeMicroRNAs(miRNAs)are small single-stranded RNA molecules with a length of about 22 nucleotides that are widely found in living organisms.In recent years,studies have found that miRNAs play important roles in osteogenic differentiation and bone formation.It has been reported that miR-92b-3p can promote osteogenic differentiation of human umbilical cord mesenchymal stem cells,but the role and mechanism of miR-92b-3p in mouse osteoblastic cell line MC3T3-E1 have not been reported.BMP-2 is an important subtype of bone morphogenetic proteins(BMPs)family,which can promote osteogenesis by regulating TGF-β/BMP signaling pathways.The purpose of this study was to investigate the role of miR-92b-3p in osteogenic differentiation of MC3T3-E1 cells induced by BMP-2 and its possible mechanism.Methods1 Effect of miR-92b-3p on osteogenic differentiation of MC3T3-E1 cells(1)MC3T3-E1 cells were cultured in complete medium or medium with BMP-2.Expression of miR-92b-3p was detected by RT-qPCR before and after induction,and ALP activity was detected by alkaline phosphatase(ALP)staining.(2)miR-92b-3p mimic or inhibitor was transfected into MC3T3-E1 cells,overexpression,interference efficiency and expression of ALP were detected by RT-qPCR,and ALP activity was detected by ALP staining.2 Study of the mechanism of miR-92b-3p regulating osteogenic differentiation of MC3T3-E1 cells(1)star Base database and DAVID database were used to predict and analyze the downstream target genes of miR-92b-3p.(2)MC3T3-E1 cells were transfected with miR-92b-3p mimic or inhibitor,the expression of target genes was detected by RT-qPCR and western blot,and the effect of miR-92b-3p on target genes was analyzed.Results1 Effects of miR-92b-3p on osteogenic differentiation of MC3T3-E1 cells(1)Compared with the control group,MC3T3-E1 cells induced by 400ng/m L BMP-2 showed increased expression of miR-92b-3p(P<0.05)and increased activity of alkaline phosphatase(ALP).(2)After MC3T3-E1 cells were transfected with miR-92b-3p mimic,the expression level of miR-92b-3p and ALP were increased(P<0.05),the activity of ALP was increased at the same time.The expression level of miR-92b-3p and ALP decreased(P<0.05)after MC3T3-E1 cells were transfected with miR-92b-3p inhibitor,and the activity of ALP decreased,too.2 Study on the mechanism of miR-92b-3p regulating osteogenic differentiation of MC3T3-El cells(1)204 target genes downstream of miR-92b-3p were predicted and screened through star Base database.KEGG analysis showed that the target genes were related to 10 metabolic pathways such as Fox O signaling pathway,and GO analysis showed that the function of target genes was related to the Smad protein family.Combined with enrichment analysis results and literature reports,it was found that Smad7,a target gene downstream of miR-92b-3p,was an important osteogenic negative regulator.(2)After MC3T3-E1 cells were transfected with miR-92b-3p mimic,the mRNA and protein expression levels of Smad7 were decreased(P<0.05).And the mRNA and protein expression levels of Smad7 were increased after transfection with miR-92b-3p inhibitor(P<0.05).ConclusionsmiR-92b-3p promoted the osteogenic differentiation of MC3T3-E1 cells induced by BMP-2,and miR-92b-3p inhibited the the expression of the osteogenic negative regulator Smad7,The mechanism of miR-92b-3p promoting the osteogenic differentiation of MC3T3-E1 cells may be that it inhibited the expression of osteogenic negative regulator Smad7.