miR-145靶向调控Smad3对人骨髓间充质干细胞成骨分化的影响

Effect of targeting regulation of Smad3 by miR-145 on the osteogenic differentiation of human bone marrow mesenchymal stem cells

【目的】探讨miR-145靶向调控Smad3对人骨髓间充质干细胞成骨分化的影响。【方法】骨髓间充质干细胞诱导分化0、3、7、10、14 d后,碱性磷酸酶(alkaline phosphatase,ALP)试剂盒检测ALP活性;Western blotting检测Runx2、osteopontin、Smad3的蛋白表达;RT-PCR检测miR-145的表达;miR-control、miR-145 mimics、anti-miR-control、miR-145 inhibitors及pc DNA-control和pc DNA-Smad3转染h BMMSCs,并进行miR-NC+miR-145 mimics、Wt-Smad3+miR-145 mimics、Mut-Smad3+miR-143 mimics3组共转染,检测ALP活性及Runx2、osteopontin、Smad3的蛋白表达。【结果】随着时间的延长,ALP活性及Runx2和osteopontin的蛋白表达升高,与0 d比较差异具有统计学意义(P<0.01);Smad3是miR-145的靶基因;随着时间延长miR-145的表达降低,而Smad3表达升高,与0 d比较差异具有统计学意义(P<0.01);miR-145 inhibitors组ALP活性及Runx2和osteopontin的蛋白表达显著高于anti-miR-control,pc DNA-Smad3组ALP活性及Runx2和osteopontin的蛋白表达显著高于pc DNA-control组(P<0.01)。【结论】miR-145可靶向调控Smad3促进人骨髓间充质干细胞的成骨分化。

【Objective】To explore the effect of targeting regulation of Smad3 by miR-145 on the osteogenic differentiation of human bone marrow mesenchymal stem cells（h BMMSCs）.【Methods】When the inducing differentiation of h BMMSCs was performed for 0, 3,7, 10 and 14 d, alkaline phosphatase（ALP） activity was detected by ALP kit, the protein expressions of Runx2, osteopontin and Smad3 were determined by Western blotting, and the expression of miR-145 was detected by RT-PCR. The h BMMSCs were transfected with miR-control, miR-145 mimics, anti-miR-control, miR-145 inhibitors, pc DNA-control and pc DNA-Smad3. There were 3 groups of cotransfection, including miR-NC＋miR-145 mimics, Wt-Smad3＋miR-145 mimics and Mut-Smad3＋miR-143 mimics. The ALP activity and protein expressions of Runx2, osteopontin and Smad3 were detected.【Results】The ALP activity and the protein expressions of Runx2 and osteopontin increased with time. Compared with those on 0 d, there was a statistically significant difference（P〈0.01）. Smad3 was the target gene of miR-145. The expression of miR-145 decreased with time, while the expression of Smad3 increased. There was a significant difference compared with those on 0 d（P〈0.01）. The ALP activity and protein expressions of Runx2 and osteopontin in the groups of miR-145 inhibitors were significantly higher than those in the anti-miR-control group. The ALP activity and protein expressions of Runx2 and osteopontin in the pc DNA-Smad3 group were significantly higher than those in the pc DNA-control group（P〈0.01）.【Conclusion】miR-145 can target Smad3 and promote the osteogenic differentiation of h BMMSCs.