慢病毒介异的孕烷X受体sh-RNA稳定干扰HepG2细胞株的构建

doi:10.3969/j.issn.1004-616x.2014.03.011

癌变·畸变·突变

慢病毒介异的孕烷X受体sh-RNA稳定干扰HepG2细胞株的构建

牛永东^1，2，陈理明³，吴曼鹏⁴，程訸¹，徐涵¹，高分飞¹，石刚刚^1，*

汕头大学医学院药理教研室，广东汕头 515041

收稿日期:2014-02-21 修回日期:2014-04-11
通讯作者: 石刚刚，E-mail：ggshi@stu.edu.cn
作者简介:牛永东(1974- )，男，山西省长治市人，博士，讲师，研究方向：肿瘤病因学。E-mail：ydniu@126.com
基金资助:
中国博士后科学基金(2012M510200)，广东省自然科学基金 (S201 2010010955)，广东省医学科研基金(A2013656)，中央财政支持地方高校发展专项资金

Establishment of a HepG2 cell line with stable PXR silencing by lentivirus-mediated RNA interference

NIU Yong-dong^1，2，CHEN Li-ming³，WU Man-peng⁴，CHENG He¹，XU Han¹，GAO Fen-fei¹，SHI Gang-gang^1，*

Department of Pharmacology, Shantou University Medical College, Shantou 515041

Received:2014-02-21 Revised:2014-04-11

摘要/Abstract

摘要：

目的：构建慢病毒介导的人孕烷X受体(hPXR)sh-RNA干扰载体及筛选肝癌细胞稳定干扰株。方法：针对hPXR的特异性序列，设计干扰序列，构建重组克隆。转染HEK293T细胞，筛选有效干扰片段。HEK293T包装干扰病毒并转染肝癌HepG2细胞株，200 mmol/L嘌呤霉素筛选2－3周，Western blot检测PXR表达。hPXR激动剂利福平处理PXR sh-RNA稳定干扰肝癌HepG2细胞株和Scramble对照细胞，抽提RNA并鉴定hPXR靶基因细胞色素P450 3A4(CYP3A4)的表达。结果：筛选出了PXR有效干扰片段，并获得了PXR信号通路有效敲低的HepG2慢病毒稳定干扰株。结论：成功构建了慢病毒介导的PXR sh-RNA稳定干扰HepG2细胞株。

关键词: 人孕烷X受体, 原发性肝细胞癌, 慢病毒, RNA干扰

Abstract:

OBJECTIVE: To establish a HepG2 cell line with stable PXR silencing effect by a lentiviral vector carrying a short hairpin RNA (sh-RNA). METHODS：Three double-stranded sh-RNA targeting the PXR gene were designed，synthesized and cloned into the psi-sH1 sh-RNA vector. The effective recombinant sh-RNA expression plasmid against PXR，which was selected of HEK293T cells with three double-stranded sh-RNA plasmids，was packaged into HEK293T cells and used to infect HepG2 cells after 200 mmol/L puromycin screening for 2-3 weeks. The expression level of PXR was detected by western blot，the CYP3A4 mRNA levels both in sh-RNA PXR HepG2 stable cells and scramble control HepG2 cells were detected by real-time PCR. RESULTS：A recombinant sh-RNA expression plasmid against PXR gene with effective interference was successfully selected and a HepG2 cell line stably transfected with the sh-RNA PXR was established. CONCLUSION：A HepG2 cell line stably transfected with the sh-RNA PXR was established.

Key words: human pregnane X receptor, hepatocellular carcinoma, lentivirus, RNA interference

牛永东，陈理明，吴曼鹏，程訸，徐涵，高分飞，石刚刚. 慢病毒介异的孕烷X受体sh-RNA稳定干扰HepG2细胞株的构建[J]. 癌变·畸变·突变, doi: 10.3969/j.issn.1004-616x.2014.03.011.

NIU Yong-dong，CHEN Li-ming，WU Man-peng，CHENG He，XU Han，GAO Fen-fei，SHI Gang-gang. Establishment of a HepG2 cell line with stable PXR silencing by lentivirus-mediated RNA interference[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2014.03.011.

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