[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、mTOR、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。

[Abstract] 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, GSK3b, MKK6, mTOR, P38, P53, P70S6K 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.

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）