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

doi:10.19586/j.2095-2341.2022.0197

生物技术进展 ›› 2023, Vol. 13 ›› Issue (2): 273-281.DOI: 10.19586/j.2095-2341.2022.0197

• 研究论文 • 上一篇

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

孙霄麟(), 张迪迪, 王馨雅, 张丽娜()

北京工业大学环境与生命学部，北京 100124

收稿日期:2022-11-16 接受日期:2022-12-16 出版日期:2023-03-25 发布日期:2023-04-07
通讯作者: 张丽娜
作者简介:孙霄麟 E-mail：sunxiaolin@emails.bjut.edu.cn；
基金资助:
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)

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

Xiaolin SUN(), Didi ZHANG, Xinya WANG, Lina ZHANG()

Faculty of Environment and Life，Beijing University of Technology，Beijing 100124，China

Received:2022-11-16 Accepted:2022-12-16 Online:2023-03-25 Published:2023-04-07
Contact: Lina ZHANG

摘要/Abstract

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pEGFP-TOE1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。

关键词: TOE1, 稳转细胞系, 慢病毒载体, SGC-7901细胞, 细胞定位

Abstract:

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.

Key words: TOE1, stable cell line, lentiviral vector, SGC-7901 cells, cellular localization

中图分类号:

Q279

孙霄麟, 张迪迪, 王馨雅, 张丽娜. 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[J]. 生物技术进展, 2023, 13(2): 273-281.

Xiaolin SUN, Didi ZHANG, Xinya WANG, Lina ZHANG. Construction of Stable Cell Lines with TOE1 Knockdown and Overexpression and Analysis on its Cellular Localization in Gastric Cancer Cells[J]. Current Biotechnology, 2023, 13(2): 273-281.

图/表 7

图1 pLVX-shTOE1和pCDH-TOE1慢病毒载体构建示意图A：敲低TOE1基因的pLVX-shTOE1慢病毒载体构建示意图；B：过表达TOE1基因的pCDH-TOE1慢病毒载体构建示意图

Fig. 1 Schematic diagram of pLVX-shTOE1 and pCDH-TOE1 lentiviral vector construction

图2 pLVX-shTOE1慢病毒载体测序比对结果

Fig. 2 Sequencing comparison results of pLVX-shTOE1 lentiviral vector

图3 TOE1基因PCR扩增、电泳鉴定及测序比对结果A：TOE1基因PCR扩增产物的琼脂糖凝胶电泳结果。M—2 000 bp DNA marker； 1—TOE1基因扩增产物。B：TOE1基因过表达pCDH-TOE1慢病毒载体菌落PCR鉴定结果。M—200 bp DNA ladder； 1~6—菌落PCR产物的琼脂糖凝胶电泳结果；C：pCDH-TOE1慢病毒载体测序结果和TOE1 cDNA序列的比对结果。

Fig. 3 PCR amplification, electrophoretic identification and sequencing comparison results of TOE1 gene

图4 敲低TOE1基因的SGC-7901稳转细胞系的荧光图（×10）

Fig. 4 Fluorescence results of SGC-7901 stable cell line with TOE1 gene knockdown (×10)

图5 TOE1在稳转胃癌细胞系中mRNA的表达水平A：RT-qPCR检测SGC-7901稳转细胞系中TOE1的敲低效率；B：RT-qPCR检测SGC-7901稳转细胞系中TOE1的过表达效率。Mock—SGC-7901空白细胞，shNC—敲低实验的阴性对照，shTOE1—敲低TOE1的SGC-7901细胞，Control—过表达实验的阴性对照，oeTOE1—过表达TOE1的SGC-7901细胞。*和**表示分别在P<0.05和P<0.01水平上差异具有统计学意义

Fig. 5 Expression of TOE1 mRNA in gastric cancer stable cell lines

图6 TOE1在稳转胃癌细胞系中蛋白的表达A：Western blot检测SGC-7901稳转细胞系中TOE1的敲低效率；B：Western blot检测SGC-7901稳转细胞系中TOE1的过表达效率。Mock—SGC-7901空白细胞，shNC—敲低实验的阴性对照，shTOE1—敲低TOE1的SGC-7901细胞，Control—过表达实验的阴性对照，oeTOE1—过表达TOE1的SGC-7901细胞。**表示在P<0.01水平上差异具有统计学意义。

Fig. 6 Expression of TOE1 protein in gastric cancer stable cell lines

图7 TOE1在SGC-7901细胞中的定位（×100）注：SGC-7901细胞中转染pEGFP-C3对照质粒和pEGFP-TOE1重组质粒后绿色荧光分布图，DAPI染细胞核呈现蓝色。

Fig. 7 Subcellular localization of TOE1 in SGC-7901 cells (×100)

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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

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