miR-210-5p对小鼠脂肪干细胞成骨分化的影响及作用机制分析

Effect and mechanism analysis of miR-210-5p on osteogenic differentiation of mouse adipose stem cells

[目的]观察miR-210-5p在小鼠脂肪干细胞增殖和成骨分化中的作用并探讨其作用机制.[方法]将小鼠脂肪干细胞分空白对照组、miR-210-5p模拟物阴性对照组、miR-210-5p模拟物组、miR-210-5p抑制物阴性对照组和miR-210-5p抑制物组,采用CCK-8细胞计数法检测细胞增殖能力,茜素红染色观察钙盐矿化结节形成能力,用实时荧光定量PCR检测miR-210-5p水平及成骨分化标志物BMP2、Smad1、Smad5和Runx2 mRNA表达水平,进而用蛋白免疫印迹法检测BMP2、Smad1、Smad5和Runx2蛋白表达水平.[结果] miR-210-5p模拟物组细胞增殖活力显著升高(P<0.01),矿化结节显著增加,BMP2、Smad1、Smad5和Runx2 mRNA和蛋白表达水平显著上调(P<0.01);miR-210-5p抑制物组细胞增殖活力显著降低(P<0.01),矿化结节显著降低,BMP2、Smad1、Smad5和Runx2 mRNA和蛋白表达水平显著下调(P<0.01).[结论] miR-210-5p可能通过调控BMP2/Smad信号通路促进小鼠脂肪干细胞增殖和成骨分化.

[Objective] In postmenopausal osteoporosis(PMOP),adipose-derived mesenchymal stem cells(ADSCs) are unbalanced in osteogenic adipogenic differentiation,resulting in a significantly weaken osteogenic differentiation capacity.Therefore,exploring mechanisms to promote osteogenic differentiation in PMOP treatment is of great significance,and microRNA is widely involved in the regulation of bone metabolism such as osteogenesis and osteoclastic differentiation.Some studies have found the down-regulation of miR-210-3p in PMOP patients and the up-regulation of miR-210 during osteogenic differentiation.However,the role of miR-210 in ADSCs has not been reported yet.This study aims to explore its effect on the osteogenic differentiation of ADSCs by knocking down and overexpressing miR-210-5p,to observe the role of miR-210-5p in the proliferation and osteogenic differentiation of mouse adipose stem cells,and to explore the mechanism.[Methods] Mouse adipose stem cells were divided into blank control,miR-210-5p mimic NC,miR-210-5p mimic,miR-210-5p inhibitor NC,and miR-210-5p inhibitor groups.Cell proliferation ability was detected using cell counting kit-8(CCK-8),and the ability to form calcium mineralization nodules was observed through Alizarin Red staining.The levels of miR-210-5p and the mRNA expression levels of osteogenic differentiation markers BMP2,Smad1,Smad5 and Runx2 were detected using real-time PCR.Subsequently,the protein expression levels of BMP2,Smad1,Smad5 and Runx2 were detected through Western blotting.[Results] The transfection efficiency of miR-210-5p was detected using real-time PCR.There was no significant difference in the expression of miR-210-5p in the miR-210-5p inhibitor NC and miR-210-5p mimic NC groups compared to the blank control group.However,the transfection of miR-210-5p inhibitor and miR-210-5p mimic could significantly inhibit and promote the expressions of miR-210-5p in ADSCs(P<0.001,P<0.01),respectively.According to the CCK-8 results,there was no significant difference in proliferation activity in the expression of miR-210-5p in the miR-210-5p inhibitor NC and miR-210-5p mimic NC groups compared to the blank control group.However,compared to the miR-210-5p mimic NC group,the proliferation activity of miR-210-5p mimic cells was significantly increased(P<0.01).According to the Alizarin Red staining results,calcium deposition in the miR-210-5p mimic group was the most abundant,indicating a significant increase in mineralization nodules(P<0.001).Moreover,real-time PCR and Western blotting results showed that the mRNA and protein expression levels of BMP2,Smad1 and Runx2 were significantly upregulated(P<0.05).Conversely,compared to the miR-210-5p inhibitor NC group,the proliferation activity of miR-210-5p inhibitor cells was significantly reduced(P<0.01),mineralization nodules were significantly reduced(P<0.001),and the mRNA and protein expression levels of BMP2,Smad1,Smad5 and Runx2 were significantly downregulated(P<0.05).[Conclusion] The BMP2/Smad pathway plays a crucial role in osteogenic differentiation of stem cells.BMP2,a member of transforming growth factor β superfamily,is the first essential growth factor to discover osteogenic differentiation.It binds to cell membrane receptors BMPR1 and BMPR2,promotes Smad1/5/8 phosphorylation,and combines with the universal signal transduction protein Smad4 to form Smad complex,which promotes Runx2 expression after nucleation.Runx2 is a key upstream transcription factor for osteogenic differentiation,which is also an early marker of osteogenic differentiation.In this study,the effects of miR-210-5p on genes and proteins involved in osteogenic differentiation in mouse ADSCs are examined.The results show that the silencing of miR-210-5p downregulates the expression levels of BMP2,Smad1,Smad5 and Runx2,while the overexpression of miR-210-5p significantly upregulates their expression levels.In conclusion,miR-210-5p may promote the osteogenic differentiation of mouse ADSCs by activating the BMP2/Smad signaling pathway.