Over-expression of miR-217 regulates EZH2 to promote osteogenic differentiation of BM-MSCs in osteoporotic mouse models

Abstract

Objective To explore the effect of microRNA-217 (miR-217) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) in osteoporosis (OP) mouse model and potential regulatory mechanism. Methods The mice were divided into OP group and sham operation (sham) group with 12 in each group and both underwent bilateral oophorectomy. The OP mouse BM-MSCs were divided into control group, miR-NC group, miR-217mimics group, miR-217 mimics+pcDNA 3.1 group and miR-217 mimics+pcDNA 3.1-EZH2(enhancer of zeste homologe 2) group. The different groups were treated by transfection with miR-NC, miR-217 mimics, miR-217 mimics plus pcDNA 3.1, miR-217 mimics plus pcDNA 3.1-EZH2 respectively and then followed by osteogenic differentiation. The mRNA and protein expressions of miR-217, EZH2, OCN, Runx2, and collagen Ⅰ were detected by RT-qPCR/Western blot; The alkaline phosphatase (ALP) activity of BM-MSCs in each group was detected by microplate reader; Alizarin red S (ARS) staining assay was used to stain the BM-MSCs in each group for microscopy; The targeting relationship between miR-217 and EZH2 was verified by the dual luciferase reporter gene. Results The expression level of miR-217 in the bone tissue and BM-MSCs of the OP group was lower than that of the sham group (P＜0.05), and the expression level of EZH2 mRNA was higher than that of the sham group(P＜0.05). The expression level of miR-217 in BM-MSCs, the expression levels of OCN, Runx2, collagen Ⅰ mRNAs and proteins, and the ALP activity in the miR-217 mimics group were higher than those in the control group and in miR-NC group (P＜0.05). The expression level of EZH2 mRNA and protein were lower than those of the control group and miR-NC group (P＜0.05). And the degree of ARS staining was heavier than that of the control group and miR-NC group. The expression levels of EZH2 mRNA and protein in BM-MSCs in the miR-217 mimics+pcDNA 3.1-EZH2 group were higher than those in the miR-217 mimics group and miR-217 mimics+pcDNA 3.1 group (P＜0.05). And the expression levels of OCN, Runx2, collagen Ⅰ mRNAs and proteins, and ALP activity were lower than those in miR-217 mimics group and miR-217 mimics+pcDNA 3.1 group (P＜0.05). The degree of ARS staining was lighter than that in miR-217 mimics group and miR-217 mimics+pcDNA 3.1 group. There were binding sites between miR-217 and the 3′UTR region of EZH2 mRNA. The relative luciferase activity of WT-EZH2+miR-217 mimics group was lower than that of WT-EZH2+miR-NC group (P＜0.05). Conclusions Over-expression of miR-217 can inhibit the expression of EZH2 and promote the osteogenic differentiation of BM-MSCs in OP mice, which could provide a new strategy for clinical prevention and treatment of OP.

Keywords

• microrna-217| enhancer of zeste homolog 2(ezh2)| bone marrow derived mesenchymal stem cell(bm-msc)| osteoporosis