摘要
目的:探讨ezrin增强子敲除对食管癌Eca-109细胞ezrin基因表达、细胞增殖和迁移的影响。方法:将靶向ezrin增强子上、下游的CRISPR/Cas9重组质粒共转染食管癌Eca-109细胞,经嘌呤霉素筛选,获得敲除ezrin增强子的细胞株Eca-C2。用qPCR和Western blotting分别检测敲除ezrin增强子的Eca-C2细胞中ezrin mRNA和蛋白的表达,用蛋白芯片技术检测MAPK通路相关蛋白的表达,用WST-1法和细胞划痕愈合实验分别检测ezrin增强子敲除对Eca-C2细胞增殖和迁移能力的影响。结果:成功构建稳定敲除ezrin增强子的食管癌细胞株Eca-C2;与对照细胞相比,ezrin增强子敲除细胞ezrin mRNA和蛋白的表达水平均明显降低(均P<0.05)。Eca-C2细胞中17种被检测的MAPK通路相关蛋白中有9种(AKT、CREB、GSK3b、MKK6、m TOR、P38、P53、P70S6K和RSK1)表达下调,ezrin增强子敲除后细胞的增殖和迁移能力受到明显抑制(均P<0.05)。结论:在人食管癌Eca-109细胞中,敲除ezrin增强子可明显抑制细胞的增殖和迁移。
Objective: To investigate the effects of ezrin enhancer knockout on ezrin gene expression, cell proliferation and migration of human esophageal carcinoma Eca-109 cells. Methods: The CRISPR/Cas9 recombinant plasmids targeting upstream/downstream of human ezrin enhancer were co-transfected into human esophageal carcinoma Eca-109 cells, and the cell line Eca-C2 with ezrin enhancer knockout was screened by purinomycin. Then the expression levels of ezrin mRNA and protein in Eca-C2 cells were detected by Real-time quantitative PCR(qPCR) and Western blotting, respectively;The expression levels of MAPK-pathway-related proteins were detected by protein array technology;and the effects of ezrin enhancer knockout on the proliferation and migration of Eca-C2 cells were analyzed by WST-1 method and wound-healing assay, respectively. Results: The human esophageal carcinoma cell line Eca-C2 with stable ezrin enhancer knockout was established successfully. Compared with control cells, the mRNA and protein expressions of ezrin in Eca-C2 cells were significantly reduced(all P<0.05). Among the 17 detected MAPK pathway related proteins in Eca-C2 cells, 9 proteins(AKT, CREB, GSK3 b, MKK6, m TOR, P38, P53, P70 S6 K and RSK1) were down-regulated, and the cell proliferation and migration were significantly inhibited(all P<0.05). Conclusion: ezrin enhancer knockout can significantly inhibit the cell proliferation and migration of human esophageal carcinoma Eca-109 cells.
作者
雷悦
野庆松
卫金岐
李文娜
莫镇涛
张青峰
高书颖
LEI Yue;YE Qingsong;WEI Jinqi;LI Wenna;MO Zhentao;ZHANG Qingfeng;GAO Shuying(Department of Biochemistry and Molecular Biology,Zhnhai Campus of Zunyi Medical University,Zhnhai 519041,Guangdong;Zhuhai Premier-Discipline Enhancement Scheme of Pharmacology,Zhnhai Campus of Zunyi Medical University,Zhnhai 519041,Guangdong,China;Department of Gastroenterology,the Fifth Affiliated Hospital of Sun Yat-sen University,Zhnhai 519000,Guangdong,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2019年第1期29-35,共7页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No.31360212
No.81760697
No.81760723)
贵州省科学技术基金(黔科合J字[2014]2180号)~~
