p62基因敲除后食管鳞状细胞癌EC109细胞生物学行为变化  

Biological behavior of p62 gene knock-out esophageal squamous cell carcinoma EC109 cells

作　　者：陈秀英 于莉 周君阳 丁妍[3] 谭玉洁[1,4] CHEN Xiuying;YU Li;ZHOU Junyang;DING Yan;TAN Yujie(Guizhou Medical University,Guiyang 550004,China)

机构地区：[1]贵州医科大学,贵阳550004 [2]三峡大学 [3]湖北医药学院胚胎干细胞研究湖北省重点实验室 [4]贵州医科大学附属医院

出　　处：《山东医药》2020年第2期10-13,21,共5页Shandong Medical Journal

摘　　要：目的观察p62基因敲除后对食管鳞状细胞癌EC109细胞生物学行为的影响。方法使用CRISPR/Cas9技术构建p62敲除的EC109细胞株作为敲除组,选择野生型EC109细胞作为对照组。采用T7E1核酸内切酶检测打靶效果,Western blotting法检测细胞p62蛋白,倒置显微镜下观察细胞形态变化,实时无标记动态细胞分析技术观察细胞增殖能力,细胞划痕实验观察细胞的迁移能力,流式细胞仪检测细胞周期。结果T7E1酶切后,敲除组在800 bp左右有敲除后的特异性条带,对照组只在870 bp左右有一条带。敲除组p62蛋白相对表达量低于对照组,细胞合胞体数量多于对照组(P均<0.01);敲除组细胞形态与对照组相比,细胞较圆且边界不清。敲除组10~80 h细胞增殖的CI值均低于同时点的对照组,细胞克隆数量少于对照组(P均<0.05);敲除组96 h划痕愈合率低于对照组,细胞周期中阻滞于G 0/G 1期的细胞比例高于对照组(P均<0.05)。结论p62基因敲除后食管鳞状细胞癌EC109细胞形成较多合胞体,且细胞的恶性行为受到抑制;p62有望成为治疗食管癌的有效基因靶点。Objective To investigate the effect of p62 gene knockout on the biological behavior of esophageal carcinoma EC109 cells.Methods We constructed the p62 knock-out EC109 cells by CRISPR/Cas9 technology,which were defined as the knockout group.At the same time,we defined wild EC109 cells as the control group.We applied T7E1 endonuclease to detect the target effect,Western blotting to detect p62 protein expression in EC109 cells,and we observed the cell morphology changes under an inverted microscope.We detected the proliferation ability of EC109 cells by the real-time cellular analysis(RTCA).We monitored the invasion ability of EC109 cells by scratch experiment and the cell cycle by flow cytometry.Results After T7E1 enzyme digestion,the knockout group had a specific band at about 800 bp,while the control group had only one band at about 870 bp.The relative expression of p62 protein in the knockout group was lower than that in the control group,and the number of cell syncytia was higher than that in the control group(P<0.01).Compared with the control group,the cells in the knockout group were round with unclear boundary.The CI value of cell proliferation in the knockout group was lower than that of the control group,the number of cell clones was less than that of the control group,the wound healing rate in the knockout group at 96 h was lower than that in the control group,and the proportion of cells blocked in the G 0/G 1 phase in the cell cycle was higher than that in the control group(all P<0.05).Conclusion Esophageal squamous carcinoma EC109 cells with p62 gene knockout can form more syncytia,which can inhibit the malignant behavior of EC109 cells,and p62 is expected to be an effective genetic target for esophageal cancer.

关 键 词：食管癌 p62基因 基因敲除 细胞形态 细胞增殖 细胞迁移 细胞周期 

分 类 号：R735.1[医药卫生—肿瘤]