RNAi介导的CK2α基因沉默对喉癌细胞上皮间质转化过程的影响

摘要

目的 探讨人喉癌Hep-2细胞稳定转染干扰质粒后,蛋白激酶CK2α表达对其上皮间质转化过程的影响.方法 应用体外构建的干扰质粒pGCsi-H1-CK2α稳定转染人喉癌上皮细胞Hep-2,分为未转染组(parental)、对照质粒转染组(pGCsi-H1-control)和干扰质粒转染组(pGCsi-H1-CK2α).转染24 h后经G418筛选出稳定转染的细胞系,应用Real-time PCR和Western blot进行鉴定;采用Transwell小室检测转染后Hep-2细胞的侵袭和迁移能力;使用相差显微镜观察各组细胞形态并通过Western blot检测细胞上皮标志物E-cadherin、vimentin及转录因子slug、snail的表达.结果 干扰质粒转染组中CK2α基因在mRNA和蛋白水平的表达明显低于未转染组及对照质粒转染组(P<0.01).Transwell实验显示稳定敲低CK2α阻止了Hep-2细胞的迁移与侵袭.与未转染组及对照质粒转染组相比,干扰质粒转染组中的E-cadherin的表达增加,但是snail、slug和vimentin的表达下降.结论 RNAi介导的CK2α抑制表达抑制了喉癌细胞上皮间质转化过程.%Objective To investigate the effect of protein kinase CK2α expression on the transformation of epithelium in human laryngeal carcinoma Hep-2 cells.Methods HepG 2 cells were transfected into human laryn-geal squamous cell carcinoma cell line Hep-2 by immunization with pGCsi-H1-CK2α in vitro and divided into un-transfected group(pGCsi-H1-control)and interfering plasmid(PGCsi-H1-CK2α).The cells were transfected with G418 for 24 h and identified by Real-time PCR and Western blot.The invasion and migration ability of Hep-2 cells were detected by Transwell chamber.The patients were observed by phase contrast microscope the expression of E-cadherin,vimentin and transcription factors slug and snail were detected by Western blot. Results The expression of CK2α gene in the transfected group was significantly lower than that in the untransfected group and control plas-mid(P<0.01).Transwell experiments showed that stable knockout of CK2α prevented the migration and invasion of Hep-2 cells.Compared with untransfected and control plasmid transfection groups,the expression of E-cadherin in the interfering plasmid transfection group increased,but the expression of snail,slug and vimentin decreased. Conclusions RNAi mediated inhibition of CK2α inhibits the transformation of epithelial mesenchymal cells in la-ryngeal squamous cell carcinoma.