miR-1-3p/206/613对LXRα和OATP1B1蛋白表达的影响及其机制研究

doi:10.12092/j.issn.1009-2501.2018.08.002

中国临床药理学与治疗学 ›› 2018, Vol. 23 ›› Issue (8): 846-854.doi: 10.12092/j.issn.1009-2501.2018.08.002

miR-1-3p/206/613对LXRα和OATP1B1蛋白表达的影响及其机制研究

叶容芳，刘名义，许玉，张艳丽，朱丹丹，丁薇，熊玉卿

南昌大学临床药理研究所，南昌 330006, 江西

收稿日期:2018-03-06 修回日期:2018-07-04 出版日期:2018-08-26 发布日期:2018-08-28
通讯作者: 熊玉卿，女，教授，博士生导师，主要从事临床药理学与药代动力研究。 Tel：0791-86360654 E-mail：xyqh2629@163.com
作者简介:叶容芳，女，硕士研究生，主要从事临床药理学的相关研究。 Tel：15797727835 E-mail：809309547@qq.com
基金资助:
国家自然科学基金资助项目（81673506）

Effects of miR-1-3p/206/613 on the expression of LXRα and OATP1B1 proteins and its mechanism

YE Rongfang, LIU Mingyi, XU Yu, ZHANG Yanli, ZHU Dandan, DING Wei, XIONG Yuqing

Clinical Pharmacology Institute of Nanchang University，Nanchang 330006，Jiangxi，China

Received:2018-03-06 Revised:2018-07-04 Online:2018-08-26 Published:2018-08-28

摘要/Abstract

摘要：

目的: 探究miR-1-3p、miR-206和miR-613对肝X受体α (LXRα)和有机阴离子转运多肽1B1（OATP1B1）蛋白表达及OATP1B1转运能力的影响及其机制研究。方法:通过生物信息学数据库预测可靶向作用于LXRα和OATP1B1 mRNA 3'-UTR的miRNAs；采用RT-qPCR、Western blot方法检测miR-1-3p/206/613及LXRα和OATP1B1 蛋白水平；采用LC-MS/MS方法检测HepG2细胞中瑞舒伐他汀（RSV）的含量；应用双荧光素酶报告基因法，研究miR-1-3p/206/613影响LXRα和OATP1B1表达的分子机制。结果: 预测结果表明miR-1-3p/206/613与LXRα和OATP1B1 mRNA 3'-UTR互补配对，且具有较高的特异性、保守性及结合稳定性。与对照组相比，miR-1-3p/206/613 mimics能明显下调HepG2细胞中LXRα蛋白表达水平16.5%、16.0%、25.1%，OATP1B1蛋白30.4%、30.5%、44.4%；相反，miR-1-3p/206/613 inhibitors明显上调LXRα蛋白表达水平13.3%、13.3%、16.0%，OATP1B1蛋白25.0%、25.6%、30.4%。当RSV浓度为5、60、125 μmol/L时，miR-1-3p/206/613 mimics显著减少HepG2细胞对RSV的摄取至对照组的0.50、0.19、0.30倍，0.49、0.24、0.23倍和0.64、0.48、0.31倍;与之相反，miR-1-3p/206/613 inhibitors上调1.26、1.59、2.07倍，1.97、2.44、2.63倍，2.22、2.86、2.93倍。与对照组相比，miR-1-3p/206/613 mimics或miR-1-3p/206/613 inhibitors，pGL/LXRα-WT报告基因荧光素酶活性分别下降38.5%、32.5%、32.8%或升高137.0%、75.8%、55.7%，而pGL/LXRα-Mut报告基因荧光素酶活性未见明显改变；同上，pGL/OATP1B1-WT报告基因荧光素酶活性下降24.3%、28.9%、32.1%或升高33.5%、48.3%、61.6%，而pGL/OATP1B1-Mut报告基因荧光素酶活性也未见明显改变。结论: miR-1-3p/206/613既可通过直接靶向作用于OATP1B1 mRNA 3'-UTR而调控OATP1B1的蛋白表达和转运功能，也可通过靶向作用于LXRα mRNA 3'-UTR而发挥间接调控作用。

关键词: miR-1-3p, miR-206, miR-613, 肝X受体α, 有机阴离子转运多肽1B1

Abstract:

AIM: To explore the effect of miR-1-3p, miR-206 and miR-613 on the expression of LXRα and OATP1B1 proteins, and the effect of them on the transporting function of OATP1B1 and further to explore the molecular mechanisms underlying the above effects. METHODS: Bioinformatics databases were used to predict the potential miRNAs targeting on the 3'-UTR of LXRα and OATP1B1 mRNA; the levels of miR-1-3p/206/613,LXRα and OATP1B1 proteins were detected by RT-qPCR and Western blotting; the content of rosuvastatin in HepG2 cells was detected by LC-MS/MS; Dual-luciferase reporter assays was used to study the molecular mechanism in which miR-1-3p/206/613 affect the expression of LXRα and OATP1B1.RESULTS:The predicted results showed that miR-1-3p/206/613 has perfect complementary with 3′-UTR of LXRα and OATP1B1 mRNA, which has relatively high specificity,conservation and binding stability. Compared with the control group, miR-1-3p/206/613 mimics could significantly down-regulate LXRα protein level in HepG2 cells by 16.5%,16.0% and 25.1%, OATP1B1 protein level by 30.4%, 30.5% and 44.4%, respectively. On the contrary, miR-1-3p/206/613 inhibitors significantly upregulated LXRα protein level by 13.3%,13.3% and 16.0%,OATP1B1 protein level by 25.0%,25.6% and 30.4%. When the concentrations of RSV were 5,60 and 125 μmol/L, miR-1-3p/206/613 mimics significantly reduced the uptakes of RSV in HepG2 cells to 0.50,0.19,0.30 fold,0.49,0.24,0.23 fold,0.64,0.48,0.31 fold of the control group, respectively; In contrast,miR-1-3p/206/613 inhibitors upregulated the uptakes of RSV by 1.26,1.59,2.07 fold,1.97,2.44,2.63 fold,2.22,2.86,2.93 fold.Compared with the control group, miR-1-3p/206/613 mimics or miR-1-3p/206/613 inhibitors decreased the luciferase activities of pGL/LXRα-WT reporter gene by 38.5%,32.5%,2.8% or increased by 137.0%,75.8%,55.7%.But they had no obvious effect on the luciferase activity of pGL/LXRα-Mut reporter gene. As above, the luciferase activities of the pGL/OATP1B1-WT reporter gene decreased by 24.3%, 28.9%,32.1% or increased by 33.5%,48.3%,61.6%,respectively.But they had no obvious effect on the luciferase activity of the pGL/OATP1B1-Mut reporter gene. CONCLUSION: miR-1-3p/206/613 can regulate the expression of OATP1B1 protein and transporting function of OATP1B1 by directly targeting the 3'-UTR of OATP1B1 mRNA, and can also exert indirect regulation through targeting the 3'-UTR of LXRα mRNA.

Key words: miR-1-3p, miR-206, miR-613, LXRα, OATP1B1

中图分类号:

R965.2

叶容芳，刘名义，许玉，张艳丽，朱丹丹，丁薇，熊玉卿. miR-1-3p/206/613对LXRα和OATP1B1蛋白表达的影响及其机制研究[J]. 中国临床药理学与治疗学, 2018, 23(8): 846-854.

YE Rongfang, LIU Mingyi, XU Yu, ZHANG Yanli, ZHU Dandan, DING Wei, XIONG Yuqing. Effects of miR-1-3p/206/613 on the expression of LXRα and OATP1B1 proteins and its mechanism[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2018, 23(8): 846-854.

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miR-1-3p/206/613对LXRα和OATP1B1蛋白表达的影响及其机制研究

Effects of miR-1-3p/206/613 on the expression of LXRα and OATP1B1 proteins and its mechanism

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