Xu Yu, Hu Jinfang, Zhang Yanli, Liu Mingyi, Zhang Hong, Xia Chunhua, Xiong Yuqing
Clinical Pharmacology Institute, Nanchang University, Nanchang, China.
Department of Pharmacy, First Affiliated Hospital of Nanchang University, Nanchang, China.
Xenobiotica. 2020 Dec;50(12):1494-1500. doi: 10.1080/00498254.2020.1777484. Epub 2020 Jun 12.
OATP1B1 is an important drug transporter with a complex regulatory mechanism. In this study, we wanted to investigate how LncRNA HOTAIR regulates the expression of OATP1B1 through its action on miR-206/miR-613 in HepG2 cells. The expression level of LncRNA HOTAIR, miR-206/miR-613, and OATP1B1 mRNA was detected by RT-qPCR, and the OATP1B1 protein level was detected by Western blot. The competitive endogenous RNA mechanism was validated by bioinformatics analysis and a dual-luciferase reporter gene assay. Our results showed that over- or under-expression of LncRNA HOTAIR correspondingly significantly increased or decreased the protein level of OATP1B1 in HepG2 cells, while no significant change in OATP1B1 mRNA level was observed. In addition, the stimulatory or inhibitory effect of LncRNA HOTAIR on OATP1B1 protein expression was correspondingly reversed by miR-206/miR-613 mimic or miR-206/miR-613 inhibitor. Finally, the reporter gene assay revealed that LncRNA HOTAIR can sponge miR-206/miR-613, which breaks the binding site of miR-206/miR-613 and OATP1B1 mRNA 3'-UTR, eliminating the stimulatory effect of LncRNA HOTAIR on OATP1B1 protein. Thus, we conclude that LncRNA HOTAIR can affect the expression of OATP1B1 in HepG2 cells by sponging miR-206/miR-613, which, in turn, prevents the binding of miR-206/miR-613 and OATP1B1 mRNA 3'-UTR.
有机阴离子转运多肽1B1（OATP1B1）是一种具有复杂调控机制的重要药物转运体。在本研究中，我们想探究长链非编码RNA（LncRNA）HOTAIR如何通过其对肝癌细胞系（HepG2细胞）中微小RNA-206（miR-206）/微小RNA-613（miR-613）的作用来调控OATP1B1的表达。通过逆转录定量聚合酶链反应（RT-qPCR）检测LncRNA HOTAIR、miR-206/miR-613以及OATP1B1信使核糖核酸（mRNA）的表达水平，并用蛋白质免疫印迹法检测OATP1B1蛋白水平。通过生物信息学分析和双荧光素酶报告基因检测验证竞争性内源RNA机制。我们的结果显示，LncRNA HOTAIR的过表达或低表达相应地显著增加或降低了HepG2细胞中OATP1B1的蛋白水平，而OATP1B1 mRNA水平未观察到显著变化。此外，miR-206/miR-613模拟物或miR-206/miR-613抑制剂相应地逆转了LncRNA HOTAIR对OATP1B1蛋白表达的刺激或抑制作用。最后，报告基因检测显示LncRNA HOTAIR可以吸附miR-206/miR-613，这破坏了miR-206/miR-613与OATP1B1 mRNA 3'-非翻译区（3'-UTR）的结合位点，消除了LncRNA HOTAIR对OATP1B1蛋白的刺激作用。因此，我们得出结论，LncRNA HOTAIR可以通过吸附miR-206/miR-613来影响HepG2细胞中OATP1B1的表达，进而阻止miR-206/miR-613与OATP1B1 mRNA 3'-UTR的结合。
