干扰PRMT5表达肝癌细胞稳转细胞株的建立及鉴定  

Establishment and identification of PRMT5 transfected hepatocellular carcinoma cell line

作　　者：季云[1] 李小琴[1] 赵婷玲[1] 孙庆梅[1] 严凌花[1] 李莉[1] Xiaoqin ZHAO Tingling SUN Qingmei YAN Linghua LI Li(Department of Chemotherapy, Cancer Center, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China)

机构地区：[1]江苏大学附属医院肿瘤治疗中心化疗科,江苏镇江212001

出　　处：《中国肿瘤外科杂志》2017年第3期186-190,共5页Chinese Journal of Surgical Oncology

摘　　要：目的探讨干扰蛋白质精氨酸甲基转移酶5(PRMT5)表达肝癌细胞稳转细胞株的建立及鉴定。方法针对PRMT5设计出该基因的有效短发夹RNA(shRNA)片段,选择合适慢病毒载体,通过基因工程技术,构建出PRMT5慢病毒载体介导的shRNA重组载体,并通过PCR以及测序检测构建序列的正确性。将重组病毒载体在病毒包装细胞中大量生产后,用病毒原液感染PRMT5表达量相对较高的SMMC-7721、BEL-7402细胞,2 d后,用最低杀死浓度的嘌呤霉素进行筛选,得到克隆样生长的细胞,扩大培养后分别提取蛋白和RNA,Western印迹和q PCR检测PRMT5的表达。结果成功构建出由人源性PRMT5慢病毒载体介导的shRNA重组载体p LKO.1-sh-PRMT5,PCR及测序均证明构建序列的正确。Western印迹法和q PCR检测,p LKO.1-sh-PRMT5干扰组的PRMT5蛋白及mRNA水平明显低于p LKO.1-sh-GFP组(P<0.05),p LKO.1-sh-PRMT5载体具有良好的干扰效果。结论运用慢病毒感染并筛选出稳转细胞株,在干扰效率及经济角度均优越于瞬时转染,为今后研究PRMT5在肝癌细胞中作用功能提供了良好的科研工具。Objective To investigate the establishment and identification of a stable cell line express- ing protein arginine methyhransferase 5 （PRMT5） interference in hepatocellular carcinoma cells. Methods The effective short hairpin RNA（shRNA） fragment of the PRMT5 gene was designed, By selecting the suitable lentiviral vector, the recombinant vector of PRMT5 lentiviral vector mediated by shRNA was constructed by gene engineering technology. The correctness of the sequence was detected by PCR and sequencing. Recombinant vi- ral vectors were produced in a large number in viral packaging cells. SMMC-7721 and BEL-7402 cells with high expression of PRMT5 were infected with virus solution for 2 d. The cells presented with clone-like growth were cloned by using the minimum kill concentration of puromycin. The expression of PRMT5 was detected by West- ern blotting and qPCR after extracting the protein and RNA. Results ShRNA recombinant vector pLKO. 1-sh- PRMT5 was successfully constructed by human derived PRMT5 lentiviral vector, and the sequence of PCR was proved to be correct. Western blotting and qPCR detection showed that the expressions of PRMT5 and mRNA in the pLKO. 1-sh-PRMT5 interference group were significantly lower than those of the pLKO. 1-sh-GFP group （P 〈 0. 05）. pLKO. 1-sh-PRMT5 vector had good interference effect of PRMT5 protein. Conclusions The lenti- viral vector which used to screen the stable cell line, was superior to the transient transfection in the interferenceefficiency and economic point of view, which provided a good research tool for studying the function of PRMT5 in hepatoma cells in the future.

关 键 词：蛋白质精氨酸N-甲基转移酶 RNA 小分子干扰 慢病毒属 载体蛋白质类 蛋白质精氨酸甲基转移酶5 

分 类 号：R735.7[医药卫生—肿瘤]