[In vitro study of joint intervention of E-cad and Bmi-1 mediated by transcription activator-like effector nuclease in nasopharyngeal carcinoma].

To explore the effect of intervention of E-cadherin (E-cad) and B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) mediated by transcription activator-like effector nuclease (TALEN) on the biological behaviors of nasopharyngeal carcinoma cells. Methods: Multi-locus gene targeting vectors pUC-DS1-CMV-E-cad-2A-Neo-DS2 and pUC-DS1-Bmi-1 shRNA-Zeo-DS2 were constructed, and the E-cad and Bmi-1 targeting vectors were transferred with TALEN plasmids to CNE-2 cells individually or simultaneously. The integration of target genes were detected by PCR, the expressions of E-cad and Bmi-1 were detected by Western blot. The changes of cell proliferation were detected by cell counting kit-8 (CCK-8) assay. The cell cycle and apoptosis were detected by flow cytometry. The cell migration and invasion were detected by Transwell assay. Results: The E-cad and Bmi-1 shRNA expression elements were successfully integrated into the genome of CNE-2 cells, the protein expression level of E-cad was up-regulated, and the protein expression level of Bmi-1 was down-regulated. The intervention of E-cad and Bmi-1 didn't affect the proliferation, cell cycle and apoptosis of CNE-2 cells, but it significantly inhibited the migration and invasion ability of CNE-2 cells. Furthermore, the intervention of E-cad and Bmi-1 together significantly inhibited the migration ability of nasopharyngeal carcinoma cells compared with the intervention of E-cad or Bmi-1 alone (all P<0.01). Conclusion: The joint intervention of E-cad and Bmi-1 mediated by TALEN can effectively inhibit the migration and invasion of nasopharyngeal carcinoma cells in vitro, which may lay the preliminary experimental basis for gene therapy of human cancer.目的：检测类转录激活因子效应物核酸酶(transcription activator-like effector nuclease，TALEN)介导的E-钙黏蛋白(E-cadherin，E-cad)、B细胞特异性莫洛尼氏白血病毒插入位点1(B-lymphoma Moloney murine leukemia virus insertion region-1，Bmi-1)基因联合干预对鼻咽癌细胞生物学行为的影响。方法：构建多位点基因打靶载体pUC-DS1-E-cad-2A-Neo-DS2(以下简称E-cad打靶载体)和pUC-DS1-Bmi-1 shRNA-Zeo-DS2(以下简称Bmi-1打靶载体)，将E-cad打靶载体、Bmi-1打靶载体及佛山科学技术学院分子医学研究院前期构建的TALEN载体以不同组合转染鼻咽癌CNE-2细胞。采用PCR检测靶基因的整合，Western印迹检测靶基因蛋白表达水平变化，细胞计数试剂盒(cell counting kit-8，CCK-8)检测细胞增殖能力变化，流式细胞仪检测细胞周期和细胞凋亡变化，Transwell实验检测细胞迁移和侵袭能力变化。结果：靶基因E-cad和Bmi-1 shRNA表达元件成功整合到鼻咽癌CNE-2细胞的基因组中；转染后鼻咽癌CNE-2细胞中E-cad的蛋白表达水平明显上升，Bmi-1的蛋白表达水平明显下降；干预E-cad及Bmi-1不影响细胞的增殖、周期和凋亡，但显著抑制细胞的迁移和侵袭能力，且E-cad和Bmi-1联合干预比单独干预更能显著抑制鼻咽癌CNE-2细胞体外迁移能力(均P<0.01)。 结论：TALEN介导的E-cad和Bmi-1联合干预能有效抑制鼻咽癌CNE-2细胞的体外迁移和侵袭，可为人类癌症的基因治疗建立前期的实验基础。.