Rao Yun, Jin Gao-feng, Liu Ming-yi, Li Xin-hua, Zhang Hong, Xia Chun-hua, Xiong Yu-qing
Yao Xue Xue Bao. 2016 Dec;51(12):1858-63.
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 mRNA. The expression level of miR-206/miR-613 and OATP1B1 mRNA and protein was determined with RT-qPCR 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 mRNA and protein. The results showed that the seed sequences of miR-206/miR-613 has perfect complementary with 3’-UTR of OATP1B1 mRNA in terms of sequence specificity. The secondary structure between miR-206/ miR-613 and 3’-UTR of OATP1B1 mRNA 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 mRNA. 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 mRNA 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 mRNA.
本研究旨在探讨miR-206/miR-613对OATP1B1基因表达的影响及其机制。采用生物信息学分析预测OATP1B1 mRNA 3'-非翻译区（3'-UTR）中潜在的miRNA靶位点。分别用RT-qPCR和蛋白质免疫印迹法检测miR-206/miR-613、OATP1B1 mRNA及蛋白的表达水平。利用荧光素酶报告基因实验探究miR-206/miR-613对OATP1B1 mRNA及蛋白表达影响的具体机制。结果显示，miR-206/miR-613的种子序列在序列特异性方面与OATP1B1 mRNA的3'-UTR具有完美互补性。miR-206/miR-613与OATP1B1 mRNA的3'-UTR之间的二级结构相当稳定。过表达miR-206/miR-613可使OATP1B1蛋白水平下调24.7%、38.8%。抑制miR-206/miR-613可使表达上调25%、38.2%。然而，过表达或抑制miR-206/miR-613对OATP1B1 mRNA的表达无影响。过表达miR-206/miR-613可使p MIR/OATP1B1-WT荧光素酶报告基因的荧光素酶活性降低35%和30%。抑制miR-206/miR-613可使表达增加33.1%和32.5%。当OATP1B1 mRNA的3'-UTR中与miR-206/miR-613互补的结合位点发生突变时，过表达或抑制miR-206/miR-613对荧光素酶活性无影响。综上所述，miR-206/miR-613通过直接靶向OATP1B1 mRNA的3'-UTR在转录后水平调控OATP1B1蛋白的表达。
