UCHL1基因缺失突变A549肿瘤细胞单克隆株的建立与特性分析
张瑜¹, 石燕², 松迪¹, 张璇², 郁琳², 何亚萍², 孙兆贵^2,*
1复旦大学上海医学院，上海 200032；²上海市计划生育科学研究所，国家人口计生委计划生育药具重点实验室，上海 200032
摘要
本研究旨在探索泛素羧基末端水解酶L1 (ubiquitin C-terminal hydrolase-L1, UCHL1)对非小细胞肺癌细胞系A549细胞的作用。用CRISPR-CAS9基因编辑技术构建UCHL1基因敲除的A549细胞株，用RT-PCR和Western blot检测A549细胞中UCHL1基因敲除情况，用CCK-8检测细胞增殖能力的变化，用流式细胞仪检测细胞周期的变化，用CCK-8检测A549细胞对顺铂药物敏感性的改变，用划痕与Transwell实验检测A549细胞迁移能力的变化，用Western blot检测与A549细胞迁移有关的蛋白表达变化。结果显示，使用CRISPR-CAS9技术构建的基因移码突变导致A549细胞株UCHL1 mRNA和蛋白缺失，UCHL1基因功能缺失后A549细胞增殖和各细胞周期比例没有明显变化，但对顺铂的药物敏感性降低，迁移能力下降，Erk1/2蛋白磷酸化水平升高。以上结果提示，UCHL1基因功能缺失可导致A549细胞顺铂耐药性提高，细胞迁移能力降低，其机制可能涉及Erk1/2信号通路的激活。
关键词： UCHL1; CRISPR-CAS9; 非小细胞肺癌; 耐药性; 迁移
分类号：Q291

Establishment and characterization of A549 tumor monoclonal cell line with UCHL1 gene deletion
ZHANG Yu¹, SHI Yan², SONG Di¹, ZHANG Xuan², YU Lin², HE Ya-Ping², SUN Zhao-Gui^2,*
1Shanghai Medical College, Fudan University, Shanghai 200032, China；²National Population and Family Planning Key Laboratory of Contraceptive Drugs & Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China
Abstract
The purpose of this study was to investigate the effects of ubiquitin C-terminal hydrolase-L1 (UCHL1) on non-small cell lung cancer cell line A549. UCHL1 gene knockout A549 cell line was constructed by CRISPR-CAS9 gene editing technique. The mRNA and protein levels of UCHL1 were examined by RT-PCR and Western blot, respectively. Cell proliferation and cycles were analyzed by CCK-8 method and flow cytometry, respectively. The sensitivity of A549 cells to cisplatin was detected by CCK-8 method. Migration ability of A549 cells was detected by scratch assay and Transwell test, and p-Erk expression level was assessed by Western blot. The results showed that UCHL1 gene knockout A549 cells were successfully constructed by CRISPR-CAS9 gene editing technique. After UCHL1 gene knockout, there was no significant change in cell proliferation and cell cycle ratios in A549 cells. UCHL1 gene knockout A549 cells exhibited decreased sensitivity to cisplatin and migration activity, as well as increased p-Erk expression level. These results suggest that the loss of UCHL1 gene function may reduce the sensitivity and migration ability of A549 cells, and this effect may be related to the activation of Erk1/2 signaling pathway.
Key words: UCHL1; CRISPR-CAS9; non-small cell lung cancer; drug resistance; migration

收稿日期：2017-09-26　　录用日期：2018-03-30
通讯作者：孙兆贵　　E-mail: sunzgbio@126.com
DOI: 10.13294/j.aps.2018.0032
引用本文：
张瑜, 石燕, 松迪, 张璇, 郁琳, 何亚萍, 孙兆贵. UCHL1基因缺失突变A549肿瘤细胞单克隆株的建立与特性分析[J]. 生理学报 2018; 70 (2): 184-192.
ZHANG Yu, SHI Yan, SONG Di, ZHANG Xuan, YU Lin, HE Ya-Ping, SUN Zhao-Gui. Establishment and characterization of A549 tumor monoclonal cell line with UCHL1 gene deletion. Acta Physiol Sin 2018; 70 (2): 184-192 (in Chinese with English abstract).



PDF全文下载地址:

点我下载PDF