TOE1敲低和过表达稳转细胞系的构建及其在胃癌细胞中的定位分析  

Construction of Stable Cell Lines with TOE1 Knockdown and Overexpression and Analysis on its Cellular Localization in Gastric Cancer Cells

作　　者：孙霄麟 张迪迪 王馨雅 张丽娜[1] SUN Xiaolin;ZHANG Didi;WANG Xinya;ZHANG Lina(Faculty of Environment and Life,Beijing University of Technology,Beijing 100124,China)

机构地区：[1]北京工业大学环境与生命学部,北京100124

出　　处：《生物技术进展》2023年第2期273-281,共9页Current Biotechnology

基　　金：国家自然科学基金项目(82172969);北京市自然科学基金面上项目(5202001)。

摘　　要：TOE1(target of Egr1)是DEDD家族脱腺苷酸化酶Caf1新发现的一个同工酶。构建TOE1敲低和过表达的稳转细胞系,并分析其在细胞内的定位,可为进一步研究TOE1基因的功能提供基础。首先将靶向沉默TOE1基因的shRNA序列插入慢病毒载体pLVX-shRNA2-Puro,构建敲低TOE1基因的慢病毒载体pLVX-shTOE1。然后通过PCR扩增TOE1基因序列克隆至慢病毒载体pCDH-CMV-MCS-EF1-Puro,构建TOE1基因过表达的慢病毒载体pCDH-TOE1。同时构建pEGFPTOE1真核表达载体,转染人胃癌细胞SGC-7901,激光共聚焦显微镜观察TOE1的亚细胞定位。慢病毒包装完成后分别感染SGC-7901细胞,用嘌呤霉素筛选稳定沉默TOE1的细胞系SGC-7901-shTOE1和稳定过表达TOE1的细胞系SGC-7901-oeTOE1,接着通过RT-qPCR和Western blot技术分别验证稳转细胞系中TOE1在mRNA和蛋白水平的表达水平。鉴定结果表明,SGC-7901-shTOE1细胞中TOE1的表达量显著降低,SGC-7901-oeTOE1细胞中TOE1的表达量明显升高,提示TOE1敲低和过表达的SGC-7901胃癌稳转细胞系构建成功。激光共聚焦显微镜观察结果显示,TOE1定位在细胞核中。研究结果为后期深入探究TOE1在胃癌中的功能以及作用机制奠定了基础。TOE1(target of Egr1)is a newly discovered isoenzyme of DEDD superfamily deadenylase Caf1.Construction of its knockdown and overexpression stable cell lines,and analysis of its cellular localization can provide a basis for further research on the function of TOE1 gene.Firstly,the shRNA sequence targeting the silencing of TOE1 gene was inserted into the lentiviral vector pLVX-shRNA2-Puro to construct the lentiviral vector pLVX-shTOE1 that knocks down TOE1 gene.Then,the TOE1 gene sequence was amplified by PCR and cloned into the lentiviral vector pCDH-CMV-MCS-EF1-Puro to construct the lentiviral vector pCDH-TOE1 with overexpression of TOE1 gene.Meanwhile,the eukaryotic expression vector pEGFP-TOE1 was constructed by the same method and transfected into human gastric cancer cell SGC-7901,and the subcellular localization of TOE1 was observed by confocal microscopy.After lentivirus packaging,SGC-7901 cells were infected with pLVX-shTOE1 and pCDH-TOE1 lentivirus,and then SGC-7901-shTOE1 cell line stably silenced TOE1 gene and SGC-7901-oeTOE1 cell line stably overexpressed TOE1 gene were selected by puromycin.Next,the knockdown and overexpression efficiencies of TOE1 at mRNA and protein levels were verified by RT-qPCR and Western blot,respectively.The identification results showed that the expression of TOE1 in SGC-7901-shTOE1 cells was significantly decreased,and the expression of TOE1 in SGC-7901-oeTOE1 cells was obviously increased,suggesting that TOE1-silenced and TOE1-overexpressed SGC-7901 stable gastric cancer cell lines were successfully constructed.Also,the confocal results showed that TOE1 was localized in the nucleus.The results laied a foundation for further exploring the function and mechanism of TOE1 in gastric cancer.

关 键 词：TOE1 稳转细胞系 慢病毒载体 SGC-7901细胞 细胞定位 

分 类 号：Q279[生物学—细胞生物学]