【摘要】 目的研究葛根素作用于成骨细胞MC3T3-E1后miRNA表达的变化以及与Runx2基因表达的关系。方法 RT-PCR和western blot法检测Runx2的mRNA表达水平和蛋白表达水平;miRNA表达谱检测葛根素作用于成骨细胞后可能靶向Runx2的miRNA;Target Scan、Pic Tar等靶点预测软件预测靶向Runx2的miRNA;RT-PCR测定miRNA-204表达量;构建Runx23’UTR/突变型Runx2 3’UTR重组质粒、合成miRNA-204mimics和miRNA-204NC共转染MC3T3-E1细胞,双荧光素酶报告基因系统验证靶基因;miRNA-204mimics和miRNA-204inhibitor转染MC3T3-E1细胞,RT-PCR和western blot检测转染前后Runx2的mRNA表达量和蛋白表达量的变化。结果与空白组比较葛根素作用后Runx2的miRNA和蛋白表达均上升;miRNA-204表达水平下降;表达谱和靶点预测软件预测靶向Runx2的miRNA共同有miR-204、miR-3072-3p等;过表达miRNA-204后Runx2的蛋白表达水平下降,干扰miRNA-204表达后Runx2的蛋白水平上升。结论葛根素在成骨细胞中可通过调节靶向Runx2基因的miRNA-204来促进细胞增值分化。更多还原

【Abstract】 Objective To study the effect of puerarin on expression of miRNA and the relationship with the Runx2 expression level in MC3T3-E1 osteoblasts. Methods The mRNA and protein expression levels of Runx2 were detected using real-time quantitative PCR and Western blotting. The miRNAs which may target to Runx2 was detected using miRNA expression spectrum after addition of puerarin in osteoblasts. Target Scan and Pic Tar software were used to predict miRNAs which may target to Runx2.The expression level of miRNA-204 was detected using real-time quantitative PCR. The Rho E 3‘UTR vector and Rho E mut 3’UTR vector were constructed. miRNA-204 mimics and miRNA-204 NC were synthesized. The target gene was verified with dual luciferase report gene assay. After transfecting the MC3T3-E1 cells with miRNA-204 mimics and miRNA-204 inhibitor,the mRNA and protein expression levels of Runx2 were detected using real-time quantitative PCR and Western blotting. Results Comparing to those in blank control,the mRNA and protein expression level of Runx2 increased,but the expression of miRNA-204 decreased after addition of puerarin. miRNAs which may target to Runx2 were miR-204 and miR-3072-3p,as both predicted by miRNA expression spectrum and target predicted software. After transfection of the miRNA-204 mimics,the protein expression level of Runx2 increased. The expression decreased after transfection of the miRNA-204 inhibitor. Conclusion Puerarin promotes cell proliferation and differentiation by regulation of miRNA-204 which targets to Runx2.更多还原