MiR-532-3P Inhibits MC3T3-E1 Cell Differentiation By Down-regulating ETS1

Objective:Bone formation disorders and increased osteoclast activity are the primary pathogenesis of osteoporosis.A variety of Micro RNAs(mi RNAs)can affect bone metabolism by regulating the expression of various proteins.However,the effect of mi R-532-3p on bone metabolism has not been reported.Therefore,this study used the MC3T3-E1 pre-osteoblasts to explore the impact of mi R-532-3p on the proliferation and differentiation of osteoblasts and further explored its mechanism.Methods:According to the diagnostic criteria,we selected 20 postmenopausal patients with osteoporosis and 20 postmenopausal patients with non-osteoporosis.The expression levels of mi R-532-3p and ETS1 in the spinous process bone were measured by real-time q PCR.Ovariectomy was used to construct a postmenopausal osteoporosis model in rats,and rats were divided into an oophorectomy group and a sham operation group according to whether performed ovariectomy.Bone-quality related parameters were measured by Micro-CT.Mi R-532-3p mimic and inhibitor were used to change the level of mi R-532-3p in MC3T3-E1 cells.The effect of mi R-532-3p on the proliferation of MC3T3-E1 cells was measured using MTT assay.Real-time q PCR and Western Blotting were used to measure mi R-532-3p,ETS1 and osteogenic markers expression levels.Alkaline phosphatase staining(APS)and Alizarin red staining(ARS)were respectively used to observe the effect of mi R-532-3p on the activity of alkaline phosphatase(ALP)and the mineralization ability of MC3T3-E1 cells.The mi RNA target gene prediction database was used to predict the target genes of mi R-532-3p and was further verified by dual-luciferase reporter gene analysis.Subsequently,transfected lentiviral vectors were used to overexpress the target genes in MC3T3-E1 cells.The effects of interesting gene on osteogenic differentiation were determined by real-time q PCR,Western Blotting,APS,and ARS in MC3T3-E1 cells,and the interesting gene were verified whether involved in osteogenic differentiation regulated by mi R-532-3p.Results:MiR-532-3p was elevated in the spinous processes of patients with low BMD and femurs of rats with low BMD.And in MC3T3-E1 cells,as the induction time increases,the content of mi R-532-3p gradually decreased.The results of the MTT assay suggested that mi R-532-3p mimic could inhibit the proliferation of MC3T3-E1 cells.Real-time q PCR and Western Blotting results indicated that mi R-532-3p mimic could inhibit osteogenic markers.APS and ARS results showed that mi R-532-3p mimic could inhibit alkaline phosphatase(ALP)activity and calcium nodule formation in MC3T3-E1 cells.The 315-321 sites of the ETS1 3’ untranslated region(3’UTR)was predicted the target of mi R-532-3p.It was also found that ETS1 expression was reduced in the spinous processes of patients with low BMD and femurs of rats with low BMD.As the induction time increases,the content of ETS1 gradually increased.Lenti-ETS1 could promote the expression of osteogenic related genes,ALP activity,and calcium nodule formation.overexpression of ETS1 could partially reverse the inhibitory effect of mi R-532-3p mimic on osteogenic differentiation.Conclusion:miR-532-3p can inhibit osteogenic differentiation by down-regulating ETS1.