类转录激活因子效应物核酸酶介导的E-cad和Bmi-1基因联合干预鼻咽癌的体外研究

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

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p UC-DS1-E-cad-2ANeo-DS2(以下简称E-cad打靶载体)和p UC-DS1-Bmi-1 sh RNA-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 sh RNA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。

Objective： 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 ofnasopharyngeal carcinoma cells. Methods： Multi-locus gene targeting vectors pUC-DS 1-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 orE-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 orE-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.