miR-144-3p在大鼠骨髓间充质干细胞成骨分化过程中的表达及其靶向调控作用

Role of miR-144-3p and its target gene in regulating osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro

目的探讨在骨髓间充质干细胞成骨分化过程中,mi R-144-3p表达的生理功能和作用机制,并预测其靶基因,为成骨细胞分化的深入研究提供实验依据。方法采用大鼠骨髓间充质干细胞(BMSCs),并在体外诱导间充质干细的成骨分化,通过q RT-PCR检测成骨分化标志基因(Runx2,OCN)以及mi R-144-3p的表达;通过向BMSCs株转染mi R-144-3p mimic以及mi R-144-3p inhibitor,检测mi R-144-3p表达对BMSCs分化的影响,并通过Western blot以及q RT-PCR检测mi R-144-3p靶蛋白在RNA水平以及蛋白水平的表达变化。结果在体外诱导BMSCs成骨分化过程中,成骨分化标志基因(Runx2,OCN)的表达量逐渐升髙,而mi R-144-3p的表达量则逐渐降低,到诱导成骨第21天表达水平最低;检测各组ALP活性发现,相比于对照组mi R-144-3p mimics转染组ALP活性显著降低(P<0.05),mi R-144-3p inhibitor转染组ALP活性显著升高(P<0.05);运用多种靶基因预测数据库Target Scan、micro RNA.org和mi Randa对mi R-144-3p进行靶基因的预测分析发现,smad4最可能是mi R-144-3p的靶基因;q RT-PCR结果表明,smad4在各组中的表达水平差异无统计学意义(P>0.05);Western blot结果显示,smad4在mi R-144-3p mimics转染组表达显著下降,在mi R-144-3p inhibitor转染组则显著升高,相比于对照组差异均有统计学意义(P<0.05)。结论 mi R-144-3p参与调控大鼠BMSCs成骨分化,其抑制成骨分化的功能是通过降低smad4转录后的表达水平来实现。

Objective To investigate the role of mi R-144-3 p in regulating osteogenic differentiation of bone marrow mesenchymal stem cells and predict its target genes. Methods Rat bone marrow mesenchymal stem cells（BMSCs） with induced osteogenic differentiation were examined for the expressions of Runx2, OCN and mi R-144-3 p. The effects of transfection with a mi R-144-3 p mimic or a mi R-144-3 p inhibitor were tested on the osteogenic differentiation of the BMSCs.The changes in the expressions of the predicted target of mi R-144-3 p in the BMSCs during induced osteogenic differentiation were examined using Western blotting and q RT-PCR. Results Rat BMSCs with induced differentiation into osteoblasts exhibited a progressive increase in the expressions of Runx2 and OCN（two markers of osteogenic differentiation）, while the expression of mi R-144-3 p gradually decreased during the differentiation till reaching the lowest level at 21 days of induction.In rat BMSCs, transfection with the mi R-144-3 p mimic significantly decreased ALP activity（P0.05） wile transfection with the mi R-144-3 p inhibitor significantly increased ALP activity（P0.05） in rat BMSCs. Analysis based on mi Randa, micro RNA.org database and Target Scan suggested that Smad4 was the most likely target gene of mi R-144-3 p. The results of q RT-PCR showed no significant differences in expression levels of Smad4 among the cells with different treatments（P0.05）, while Western blotting revealed a significantly decreased expression of Smad4 in the cells transfected with mi R-144-3 p mimics and an increased Smad4 expression in the cells transfected with the mi R-144-3 p inhibitor as compared with the control cells（P0.05）.Conclusion mi R-144-3 p participates in the regulation of osteogenic differentiation of rat BMSCs, and its inhibitory effect on osteogenic differentiation is achieved probably by decreasing the expression level of Smad4.