Effect Of MiRNA-3077-5p On Jaw BMSCs Differentiation In Postmenopausal Osteoporotic Mice

Objective: To analyze the effect of microenvironment of Postmenopausal osteoporosis(POP)mice on mandibular bone marrow mesenchymal stem cells(MBMSCs).To investigate the effect of miRNA-3077-5p on the differentiation ability of MBMSCs.Methods: 1.The mouse model of postmenopausal osteoporosis was established.The mouse mandible was removed under aseptic conditions,and the attached musculofascia and front and rear teeth were removed,MBMSCs were cultured by whole pulp method and tissue block method,and cell phenotypes were identified by flow cytometry.2.S-MBMSCs of mice in the Sham surgery(SHAM)group and O-MBMSCs of mice in the ovariectomy(OVX)group were induced for osteogenesis and adipogenesis.Alizarin red staining and quantification,oil red O staining and quantification,real-time PCR(RT-qPCR)and Western blot(WB)techniques were used to analyze the effects of POP mice on the biological characteristics of MBMSCs.3.The endogenous expression level of miRNA-3077-5p in the third-generation S-MBMSCs and O-MBMSCs was detected by RT-qPCR.The osteogenic and adipogenic differentiation of S-MBMSCs was induced at 0,3 and 7 days,and the relative expression level of miRNA-3077-5p in S-MBMSCs at each induction time point was detected by RT-qPCR.The artificial synthetic miRNA-3077-5p mimics/inhibitors were used to regulate the expression of miRNA-3077-5p in S-MBMSCs positively and negatively.After osteogenic and adipogenic induction,alizarin red staining and quantitative,oil red O staining and quantitative,WB technology was used to detect the expression of osteogenic and adipogenic differentiation related genes after transfection.Results: 1.The results of Micro-CT scanning and the analysis of bone histometrics indexes all proved that the POP mouse model was established successfully.When the whole pulp method and tissue block method cultured MBMSCs to the third generation,the purity was high,and they showed findle-shaped and polygonal adherent growth.The cultured cells were identified as MBMSCs by flow cytometry.The expression of surface antigens Sca-1,CD29 and CD106 of MBMSCs was high,while the expression of CD34 and CD45 was low.2.After osteogenic induction of MBMSCs,the number and morphology of calcified nodules formed by S-MBMSCs were both stronger than those formed by O-MBMSCs,and the m RNA and protein relative expressions of osteogenic related genes RUNX2 and OCN in S-MBMSCs were higher than those in O-MBMSCs group.After adipogenic induction of MBMSCs,the number and morphology of lipid droplets formed by O-MBMSCs were both higher than those formed by S-MBMSCs,and the m RNA and protein relative expressions of lipid related genes LPL and PPAR-γ in O-MBMSCs were higher than those in S-MBMSCs group.3.The expression of miRNA-3077-5p in O-MBMSCs was significantly higher than that in S-MBMSCs.The expression level of miRNA-3077-5p decreased gradually with the extension of time after S-MBMSCs osteogenic induction 0,3 and 7 days.The expression level of miRNA-3077-5p increased gradually with the prolonging of time after S-MBMSCs adipogenic induction 0,3 and 7 days.After the chemical synthesis of miRNA-3077-5p mimics/inhibitors,the expression of miRNA-3077-5p in MBMSCs was positively and negatively regulated.After osteogenesis induction,the number and volume of calcified nodules presented by alizarin red staining were the largest in the S-MBMSCs/inhibitor group,and the protein relative expression levels of osteogenic related genes RNUX2 and OCN were the highest,followed by the S-MBMSCs /control group,S-MBMSCs/mimics group at the least.After induction of lipid formation,the S-MBMSCs /inhibitor group showed the least number and small volume of lipid droplets after oil red O staining,and the relative expression of lipid related genes LPL and PPAR-γ was the least.The S-MBMSCs/control group was stronger than the S-MBMSCs/inhibitor group,and the S-MBMSCs/mimics group was the most.Conclusion: 1.In POP induced by estrogen deficiency,the ability of osteogenesis and adipogenic differentiation of MBMSCs is changed,and the ability of osteogenesis is weakened while the ability of adipogenic differentiation is enhanced.2.In MBMSCs of POP mice,miRNA-3077-5p was overexpressed,which broke the balance of osteogenic and adipogenic differentiation of MBMSCs,decreased its ability of osteogenic differentiation,and enhanced its ability of adipogenic differentiation.