MiR-133a-3p Affects Proliferation And Differentiation Of Osteoblasts By Regulating RUNX2 In High Glucose Environment

OBJECTIVE: The proliferation and differentiation of osteoblasts are closely related to the occurrence of osteoporosis.It is well known that RUNX2 can affect the proliferation and differentiation of osteoblasts.It has been found that miR-133a-3p can target RUNX2 and affect its expression,however,it is high.Whether the expression level of miR-133a-3p changes in the sugar environment and whether it participates in the pathogenesis of diabetic osteoporosis(DOP)by affecting the proliferation and differentiation of osteoblasts remains unclear.METHODS: CCK8 assay was used to detect the proliferation of MC3T3-E1 cells at different sugar concentration and time.Mi-133a-3p and RUNX2,which are important components of bone formation regulatory network,were used as entry points to transfect and group MC3T3-E1 cells.Real-time quantitative PCR was used to detect the expression of microRNA-133a-3p and osteogenic differentiation-related genes RUNX2,Osteorix,OCN and FN in different groups.Western blot was used to detect the expression of RUNX2,Osteorix,and OCN.Confocal microscopy was used to confirm the regulation of microRNA-133a-3p on the expression of RUNX2.The activity of alkaline phosphatase(ALP)was detected by ELISA in different groups of cells.It was clarified from the cellular and molecular level whether glucose affected the expression of microRNA-133a-3p,thus regulating RUNX2 and participating in the pathogenesis of osteoporosis(OP).Results: The proliferation of MC3T3-E1 cells in high glucose environment was significantly lower than that in normal group,and the expression of microRNA-133a-3p was high in high glucose environment(P=0.001).The expression of RUNX2 decreased after over-expression of lentivirus in microRNA-133a-3p,which affected the expression of downstream molecules such as Osterix,and decreased the expression of osteoblast differentiation markers such as ALP and OCN.However,there was no significant change in cell proliferation in the over-expression group of microRNA-133a-3p compared with the negative control group.Inhibiting the expression of RUNX2,Osteorix,OCN and other proteins in MC3T3-E1 cells could be promoted by inhibiting the expression of microRNA-133a-3p,and the proliferation of MC3T3-E1 cells would be slowed down after highly differentiated.Conclusion: High glucose environment can highly express miR-133a-3p in osteoblasts,and overexpression of miR-133a-3p can inhibit the differentiation process of osteoblasts,which may be one of the mechanisms of diabetic osteoporosis,but miR-133a-3p overexpression does not slow down the proliferation of osteoblasts.The slower proliferation of osteoblasts in high glucose environment may be caused by other mechanisms.Inhibition of miR-133a-3p expression reverses the inhibition of osteoblast differentiation by high glucose environments,but cell differentiation reduces the ability to continue to proliferate.