| BACKGROUNDOsteoporosis(Osteoporosis,OP)is one of the most common clinical bone metabolic diseases with the characteristics of bone mineral density(BMD),bone mass reduction and trabecular bone structure disorder,which have a serious impact on the health and normal life of patients.Effectively stimulating osteoblast activity and increasing the number of osteoblasts can significantly improve the symptoms of osteoporosis.Explore the mechanism of osteoblast differentiation can provide a new idea for the treatment of osteoporosis.In recent years,the relationship between miRNA and bone metabolism has attracted much attention,some specific miRNA and its targets have been found to play a prominent role in the process of osteogenic differentiation.Exploring the role and mechanism of miRNA in osteogenic differentiation is helpful to provide a new theory for the treatment of bone diseases.In this study,miR-877-3p was used as the research object to explore the role and mechanism of miR-877-3p in osteogenic differentiation of MC3T3-E1 cells,which provided a potential treatment for bone formation disorders.METHODS1.Effect of miR-877-3p on the osteogenic differentiation of MC3T3-E1 cells induced by TGF-β1(1)MC3T3-E1 was treated with TGF-β1 and the expression of miR-877-3p,RUNX2,COL1A1 and OSX were detected by real-time fluorescence quantitative PCR,Western Blotting was used to detect the expression of RUNX2,COL1A1 and OSX,and alkaline phosphatase activity was also detected.(2)MC3T3-E1 cell was transfected with miR-877-3p mimics or inhibitor,and the overexpression and interference efficiency were verified by real-time fluorescence quantitative PCR.The expression of RUNX2、COL1A1和OSX were detected by real-time fluorescence quantitative PCR.The transfected cells were treated with osteogenic differentiation medium,and the formation of mineralized nodule was observed by alkaline phosphatase staining and alizarin red staining.(3)In vivo experiments verified the effect of miR-877-3p on osteogenic differentiation of MC3T3-E1 cells2.Study on the mechanism of miR-877-3p promoting the osteogenic differentiation of MC3T3-E1 cells via regulating the expression of Smad7.(1)Bioinformatics analysis was used to predict the downstream target gene of miR-877-3p,the regulatory effect of miR-877-3p on Smad7 were verified by luciferase reporting system was used to detect;(2)The expression of Smad7 in MC3T3-E1 cell was detected by real-time fluorescence quantitative PCR and Western Blotting after treatment with TGF-β1;(3)MC3T3-E1 cells was transfected with miR-877-3p mimics or inhibitor,the expression of Smad7 was detected by real-time fluorescence quantitative PCR and Western Blotting.(4)TGF-β1 was added after transfection of miR-877-3p inhibitor in MC3T3-E1 cells,the expression of Smad7,RUNX2,COL1A1 and OSX were detected by real-time fluorescence quantitative PCR.The expressions of RUNX2,Smad7,p-Smad2,t-Smad2,p-Smad3 and t-Smad3 were detected by Western Blotting.(5)miR-877-3p mimics and Smad7 overexpressed plasmids were co-transfected in MC3T3-E1 cells,after treatment with osteogenic induction medium.the expression of Smad7,RUNX2,COL1A1 and OSX were detected by real-time fluorescence quantitative PCR.ALP staining and alizarin red staining were used to observe the formation of mineralized nodules.RESULTS1.Effect of miR-877-3p on osteogenic differentiation of MC3T3-E1 cells induced by TGF-β1.(1)TGF-β1 could promote the osteogenic differentiation of MC3T3-E1 cells(2)Overexpression of miR-877-3p promoted osteogenic differentiation of MC3T3-E1 cells.Conversely,interference with miR-877-3p inhibited osteogenic differentiation of MC3T3-E1 cells(3)The overexpression of miR-877-3p was demonstrated to promote osteogenic differentiation of MC3T3-E1 cells in vivo2.Study on the mechanism of miR-877-3p promoting the osteogenic differentiation of MC3T3-E1 cells via regulating the expression of Smad7.(1)Smad7 was the downstream target gene of miR-877-3p.(2)After MC3T3-E1 cells were treated with TGF-β1,the expression of Smad7 decreased.(3)miR-877-3p inhibited the expression of Smad7 in the osteogenic differentiation of MC3T3-E1 cells(4)TGF-β1 reversed the inhibition of miR-877-3p resulting in decreased osteogenic differentiation.(5)miR-877-3p promoted osteogenic differentiation of Mc3t3-el cells by inhibiting Smad7.CONCLUSION TGF-β1 promoted the osteogenic differentiation of MC3T3-E1 cells and the expression of miR-877-3p,and miR-877-3p then promote the osteogenic differentiation of MC3T3-E1 cells.M-877-3p promoted the osteogenic differentiation of MC3T3-E1 cells by inhibiting the expression of its target gene Smad7.Down-regulation of Smad7 could improve the osteogenic differentiation ability of MC3T3-E1 cells. |
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