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探究miR-206/mi R-613对OATP1B1基因和蛋白表达的影响及其调控机制。通过生物信息学软件预测可靶向作用于OATP1B1 mRNA 3’-UTR的mi RNAs;采用RT-q PCR和Western blot等分子生物学方法检测mi R-206/mi R-613、OATP1B1 mRNA及蛋白表达水平;采用双荧光报告基因法,研究mi R-206/mi R-613作用于OATP1B1表达的确切机制。结果发现,mi R-206/mi R-613种子序列与OATP1B1 mRNA 3’-UTR互补配对,具有较高的特异性,且它们之间形成的二级结构较为稳定。与对照组相比,过表达mi R-206/mi R-613,Hep G2细胞中OATP1B1的蛋白表达水平分别下调24.7%、38.8%,反之,抑制其表达OATP1B1的蛋白表达水平分别上调25%、38.2%,而OATP1B1 m RNA表达均未发现明显改变;与对照组相比,过表达或抑制表达mi R-206/mi R-613,pMIR/OATP1B1-WT报告基因荧光素酶活性分别下降35%、30%或增加33.1%、32.5%,而在OATP1B1 m RNA3’-UTR mi R-206/miR-613结合位点突变的报告基因系统中,过表达或抑制表达mi R-206/mi R-613对荧光素酶活性均无影响。因此,mi R-206/mi R-613通过直接靶向作用于OATP1B1 mRNA 3’-UTR,从而转录后调控OATP1B1蛋白表达。
To explore the effect of miR-206 / mi R-613 on OATP1B1 gene and protein expression and its regulatory mechanism. MiRNAs targeting miRNAs 3’-UTR of OATP1B1 mRNA were predicted by bioinformatics software. The mRNA and protein expressions of mi R-206 / mi R-613 and OATP1B1 were detected by RT-q PCR and Western blot. Level. The exact mechanism by which mi R-206 / mi R-613 acts on OATP1B1 expression was studied by dual fluorescent reporter assay. As a result, it was found that the mi R-206 / mi R-613 seed sequence is paired with the 3’-UTR of OATP1B1 mRNA, has high specificity, and the secondary structure formed between them is relatively stable. Compared with the control group, OATP1B1 protein expression was down-regulated by 24.7% and 38.8% in mi R-206 / mi R-613 and Hep G2 cells, respectively. OATP1B1 protein expression was up-regulated by 25% 38.2%, while the expression of OATP1B1 m RNA did not change significantly. Compared with the control group, the luciferase activity of overexpressed or downregulated mi R-206 / mi R-613 and pMIR / OATP1B1-WT reporter genes decreased by 35% , 30% or 33.1% and 32.5%, respectively. However, in the reporter gene system with OATP1B1 m RNA3’-UTR mi R-206 / miR-613 binding site mutation, 613 had no effect on luciferase activity. Therefore, mi R-206 / mi R-613 directly regulates OATP1B1 protein expression by direct targeting to the 3’-UTR of OATP1B1 mRNA.