Basic Research On MR-30d Inhibiting Osteogenic Differentiation Of BMSCs By Targeting SOCS1

ObjectivesClinically,the repair of large-area bone defects caused by peri-implantitis,tooth extraction,and tumor surgery has always been a difficult problem.Many studies have shown that microRNAs(miRNAs)are involved in the regulation of osteogenic differentiation.Therefore,this study started from miR-30 d to explore its regulation mechanism in bone regeneration and provide new treatment ideas for the repair of bone defects.Materials and methods1.Isolation and culture of rat bone marrow stem cells(BMSCs);RT-PCR confirmed the concentration of miR-30 d inhibitor transfected BMSCs;using alkaline phosphatase staining and activity detection,alizarin red staining and semi-quantitative?RT-PCR?western blot?cck-8 and other methods to test the effect of miR-30 d inhibitor on the osteogenic differentiation and proliferation of BMSCs.2.Applying database to predict the downstream target genes of miR-30d;RT-PCR? western bolt to detect its expression level;dual luciferase report further confirmed the prediction results;transfecting si-SOCS1 into BMSCs containing miR-30 d inhibitor,RT-PCR and western blot were used to detect the differentiation of BMSCs into osteogenic direction.3.The cell morphology of BMSCs transfected with antagomir-30 d on CPC was observed by electron microscopy at 4h and 24 h respectively.The osteogenic differentiation ability of BMSCs transfected with antagomir-30 d on CPC scaffolds was detected by RT-PCR.Results1.Successful isolation of rat BMSCs;When the concentration of miR-30 d inhibitor is 200 nM,the transfection effect is appropriate;after inhibition of miR-30 d,alkaline phosphatase staining and activity detection?alizarin red staining and semi-quantitative?related osteogenic genes at mRNA and protein levels both are highly expressed,but has no obvious effect on cell proliferation.2.The database predicts that the downstream target gene of miR-30 d may be SOCS1;RT-PCR?western blot results indicate that miR-30 d negatively regulates the expression of target gene SOCS1 at the transcriptional level and protein level;dual luciferase report results further indicate that SOCS1 is the direct target gene of miR-30d;inhibition of SOCS1 attenuates the role of miR-30 d inhibitor in promoting osteogenesis and the role of miR-30 d in inhibiting osteogenesis is achieved by targeting SOCS1.3.After transfection of antagomor-30 d,electron microscopy showed that BMSCs had been attached to the CPC scaffolds,but it was not completely spread at 4 h.At 24 h,the cells were completely spread out and many branches were extended on CPC scaffolds;RT-PCR results showed a significant increase in the expression of osteogenic related genes after inhibition of miR-30 d.ConclusionInhibition of miR-30 d can effectively promote the differentiation of BMSCs into osteogenesis;It is further elucidated that miR-30 d exerts an inhibitory effect on osteogenesis by targeting SOCS1,possibly because SOCS1 is involved in a certain osteogenic pathway or immune regulation;CPC scaffolds can be used as a good cell carrier and can be used in tissue engineering complexes.The above results provide an experimental basis for the application of miR-30 d in the treatment of bone defects and promoting bone regeneration.