| BACKGROUNDTrauma and the physiologically or pathologically abnormal bone resorption may all lead to bone defects,which have already seriously threatened the health of people all over the world,but the treatment of bone defects still faces enormous challenges.In the treatment of bone defects,a large number of studies have found that microRNA-17?92 plays an important role in the regulation of biological functions,such as osteoblast proliferation,differentiation and so on.The MC3T3-E1 cells can specifically express osteogenesis-related transcription factors and early phenotypic markers of osteogenesis,such as Runx-2,alkaline phosphatase(ALP)and so on,and the cells have been widely used to study the osteogenic differentiation.However,in the study about osteogenic differentiation of MC3T3-E1 cells,whether miR-92a-1-5p could regulate osteogenic differentiation of the osteogenic cells through the specific signaling pathways,and what is the molecular mechanism of regulation,and there are still have not any relevant reports.OBJECTIVEMC3T3-E1 cells were used to investigate whether miR-92a-1-5p regulates osteogenic differentiation of MC3T3-E1 cells via Wnt/?-catenin signaling pathway,and to elucidate the molecular mechanism of regulation of miR-92a-1-5p.METHODS1.The effect of miR-92a-1-5p on osteogenic differentiation of MC3T3-E1 induced by BMP-21)The osteogenic differentiation of MC3T3-E1 cells was induced by 300 ng/ml BMP-2,and the expression levels of miR-92a-1-5p,ALP,Runx-2 and OSX were detected by qRT-PCR and Western Blot.2)MTT assay was used to detect the proliferation of MC3T3-E1 cells before and after induction.3)The miR-92a-1-5p mimics or inhibitors was transfected into MC3T3-E1 cells to build an over-expression or low-expression models,and using the qRT-PCR to detect the overexpression or interference efficiency,and then,the expression level of ALP,Runx-2 and OSX in cells were detected by qRT-PCR,and the activity of ALP in cells was detected by alkaline phosphatase kit.4)After transfection of miR-92a-1-5p mimics or inhibitor in MC3T3-E1 cells,BMP-2 was added to induce osteogenic differentiation of MC3T3-E1 cells for 7 days,and the ability of osteogenic differentiation of MC3T3-E1 cells was detected by alizarin red S staining.2.The mechanism of miR-92a-1-5p on inhibiting osteogenic differentiation of MC3T3-E1 cells by regulating ?-catenin.1)Literature investigation,Bioinformatics and online database analysis were used to predict the downstream target genes of miR-92a-1-5p.2)The miR-92a-1-5p mimics or inhibitor were transfected into MC3T3-E1 cells to construct a miR-92a-1-5p over-expression or low-expression model.The expression levels of?-catenin were detected by qRT-PCR and Western Blot.3)The miR-92a-1-5p mimics were transfected into MC3T3-E1 cells,and the Wnt/?-catenin signaling pathway inhibitor lithium chloride(Licl)was added into culture in cells for 48h,and the expression level of ?-catenin,ALP,Runx-2 and OSX were both detected by qRT-PCR.4)The miR-92a-1-5p mimics and GSK-3? siRNA were co-transfected into MC3T3-E1 cells,and the expression levels of ?-catenin and Runx-2 were detected by qRT-PCR and Western Blot,and the expression level of ALP,Runx-2 and OSX were detected by qRT-PCR.5)The miR-92a-1-5p mimics was transfected into MC3T3-E1 cells,and then added Wnt/?-catenin signaling pathway inhibitor lithium chloride(Licl)for 48h,and then added BMP-2 into MC3T3-E1 cell to induce osteogenesis.After differentiation for 7 days,the osteogenic differentiation ability of MC3T3-E1 cells was detected by Alizarin Red S staining.RESULTS1.The effect of miR-92a-1-5p on osteogenic differentiation of MC3T3-E1 induced by BMP-21)The osteogenic differentiation of MC3T3-E1 cells was successfully induced by BMP-2.The expression level of miR-92a-1-5p was gradually down-regulated with the prolongation of induction time.the expression level of mRNA of osteogenic differentiation related factors ALP,Runx-2 and OSX were gradually up-regulated,and the protein expression level of Runx-2,a key transcription factor for osteogenic differentiation,was also significantly up-regulated.2)After MTT assay,the MC3T3-E1 cells were spindle-shaped,spindle-like or polygonal,and the nuclei became larger,but the proliferation was less than that before induction.3)The miR-92a-1-5p over-expression or low-expression model was successfully constructed.After transfection of miR-92a-1-5p mimics,the expression levels of osteogenic differentiation related factors ALP,Runx-2 and OSX were significantly down-regulated.The activity of ALP was also significantly weakened.After transfection of miR-92a-1-5p inhibitor,the expression levels of osteogenic differentiation related factors ALP,Runx-2 and OSX were significantly up-regulated,and the activity of ALP was enhanced.4)When miR-92a-1-5p mimics were transfected into MC3T3-E1 cells and BMP-2 was added to induce osteogenic differentiation for 7 days,the number of mineralized nodules formed in the cells was significantly reduced,indicating that the osteogenic differentiation ability was weakened.After miR-92a-1-5p inhibitor was transfected into the cells and induced by BMP-2 for 7 days,the mineralized nodules formed in the cells were significantly increased,means that the osteogenic differentiation ability was enhanced.2.The mechanism of miR-92a-1-5p on inhibiting osteogenic differentiation of MC3T3-E1 cells by regulating ?-catenin.1)Literature investigation,Bioinformatics and online database analysis predict that miR-92a-1-5p can bind to the 3' URT region of ?-catenin,indicating that the downstream target gene of miR-92a-1-5p is(3-catenin.2)After transfected with miR-92a-1-5p mimics,the mRNA and protein levels of ?-catenin were down-regulated,and the mRNA and protein expression of ?-catenin was obviously increased after transfecting the miR-92a-1-5p inhibitor.3)When miR-92a-1-5p mimics were transfected into MC3T3-E1 cells,the expression levels of?-catenin and osteogenic differentiation-related factors ALP,Runx-2,and OSX were significantly down-regulated,while Wnt/?-catenin signals pathway inhibitor lithium chloride(Licl)were added into the cells,the ?-catenin and osteogenic differentiation-related factors ALP,Runx-2,OSX expression was significantly upregulated.The results show that Licl can partially reverse the inhibitory effect of miR-92a-1-5p on the expression of?-catenin and osteogenic differentiation related factors ALP,Runx-2 and OSX in cells.4)After co-transfection of miR-92a-1-5p mimics and si-NC in MC3T3-E1 cells,the expression levels of ?-catenin and Runx-2 in the cells were significantly down-regulated,and the osteogenic differentiation-related factors ALP,Runx-2,and OSX were also down-regulated.However,after co-transfection of miR-92a-1-5p mimics and si-GSK-3? in MC3T3-E1 cells,the expression levels of?-catenin and Runx-2 in the cells were significantly upregulated,and the osteogenic differentiation-related factors ALP,Runx-2,and OSX were also up-regulated.It is shown that just like the effect of GSK-3?,miR-92a-1-5p mimics can inhibit the expression of the downstream target gene ?-catenin.5)The miR-92a-1-5p mimics were transfected into MC3T3-E1 cells,and then added with Wnt/?-catenin signaling pathway inhibitor lithium chloride(Licl)for 48 h,and then BMP-2 was added to induce osteogenic differentiation for 7 days,and it found that the ability of osteogenic differentiation was enhanced compared with transfected miR-92a-1-5p mimics.CONCLUSIONThe miR-92a-1-5p can inhibit the expression of ?-catenin,a key protein in Wnt/?-catenin signaling pathway,and then inhibit the expression of osteogenic differentiation related factors ALP,Runx-2 and OSX,and result in the osteogenic differentiation of MC3T3-E1 cells inhibited. |
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