miR-206/miR-613对OATP1B1基因和蛋白表达的影响及其机制研究

Effect and mechanism of miR-206/miR-613 on expression of OATP1B1

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。

This study was designed to explore the effect and mechanism of miR-206/miR-613 on the expression of OATP1B1 gene. Bioinformatic analysis was used to predict the potential miRNAs target sites in 3＇-untranslated region（3＇-UTR） of OATP1B1 m RNA. The expression level of miR-206/miR-613 and OATP1B1 m RNA and protein was determined with RT-q PCR and Western blot, respectively. Luciferase assay was used to explore the exact mechanism of the effect of miR-206/miR-613 on the expression of OATP1B1 m RNA and protein. The results showed that the seed sequences of miR-206/miR-613 has perfect complementary with 3＇-UTR of OATP1B1 m RNA in terms of sequence specificity. The secondary structure between miR-206/ miR-613 and 3＇-UTR of OATP1B1 m RNA was rather stable. The OATP1B1 protein level was down-regulated by 24.7%, 38.8% by overexpression of miR-206/miR-613. The expression was up-regulated by 25%, 38.2% by inhibition of miR-206/miR-613. However, overexpression or inhibition of miR-206/miR-613 had no effect on the expression of OATP1B1 m RNA. The luciferase activity of p MIR/OATP1B1-WT luciferase reporter gene was decreased by 35% and 30% through overexpression of miR-206/miR-613. The expression was increased by 33.1% and 32.5% through inhibition of miR-206/miR-613. When the binding sites in the 3＇-UTR of OATP1B1 m RNA complementary with miR-206/miR-613 was mutated, overexpression or inhibition of miR-206/ miR-613 had no effect on the luciferase activity. Collectively, miR-206/miR-613 post-transcriptionally regulates the expression of OATP1B1 protein by directly targeting the 3＇-UTR of OATP1B1 m RNA.