Effect of estrogen on osteogenic differentiation and microRNAs expression in rat bone marrow mesenchymal stem cells

Abstract

Objective To investigate the effect of estrogen on osteogenic differentiation and the expression levels of miR-20a, miR-29a in rat bone marrow mesenchymal stem cells (BMSCs) . Methods BMSCs were isolated and cultured, and were divided into 3 groups; Blank control group was cultured in complete medium and was named CM group; Control group was cultured in osteogenic differentiation medium and was named OM group; Treatment group was cultured in osteogenic differentiation medium containing 17β-estradiol (E2) and was named E2 group. Cells′ alkaline phosphatase activities were detected after being incubated for 1, 5, 9 days and the expression levels of osteogenic related proteins such as Runt-related transcription factor 2 (RUNX2) , bone morphogenetic protein 2 (BMP2) , Osteopontin (OPN) were detected in day 5 and 9 by western blot. Expression levels of miR-20a-5p, miR-29a-3p were detected in day 5 and 9 by quantitative polymerase chain reaction (qPCR) . The production of mineralized nodule were stained by Alizarin Red in day 19. The t test was used to compare differences between 2 groups and the One-Way ANOVA was used to compare differences among more than 2 groups. P<0.05 was considered statistically significant. Results Alp activities: In day 1, the figures were low in every group and there were no significant differences between each group (CM1 d: 3.49, OM1 d: 2.82, E21 d: 2.92; F= 2.002, P= 0.216) ; In day 5, the alp activities in OM group and E2 group raised markedly (E21 d: 2.92, E25 d: 15.83; t= 5.065, P= 0.007; OM1 d: 2.82, OM5 d: 8.38; t= 13.39, P= 0.0002) ; The figure in E2 group was significantly twice as in OM group and was four times as in CM group (F= 13.95, P= 0.0055) ; In day 9, alp activities increased significantly by 3 folds in all the groups compared with the figures in day 5 (CM9 d: 14.66, OM9 d: 22.17, E29 d: 45.99; CM: t= 11.830, P= 0.0003; OM: t= 8.068, P= 0.0013; E2: t= 9.332, P= 0.0007) ; The figure in OM group was 1.5 times as in CM group and the figure in E2 group was 2 times as in OM group (F= 119.1, P 0.999, Mann-Whitney U= 10.000, 9 d: P= 0.680) . Conclusions E2 promoted the osteogenic differentiation in rat BMSCs, and the expression level of miR-20a-5p increased during this process. Key words: Estradiol; Rats; Mesenchymal stem cells; Osteogenic differentiation; MicroRNA