摘要
目的同源物2增强子(EZH2)在喉癌组织上皮-间充质转化(EMT)中的作用尚不清楚。文中旨在探讨EZH2在喉癌细胞EMT中的作用。方法根据喉癌细胞系分为:TU177、SNU46、M4E、Tu686)和人类鼻咽上皮细胞(HNPECs)NP69。选取EZH2蛋白表达较高的TU177和Tu686细胞系转染EZH2si-RNA,其中转染效果高的为EZH2-1组,转染效果较低(沉默EZH2)的为EZH2-2组,对照为转染空载体的si-NC组。通过qRT-PCR和Western blot检测喉癌组织和细胞中EZH2、分泌的卷曲相关蛋白1(SFRP1)及组蛋白H3(H3K27me3)上赖氨酸27的三甲基化的表达。转染si-EZH2、si-SFRP1、oe-SFRP1或H3K27me3上调后,通过CCK-8检测细胞活力,免疫印迹法检测E-钙粘蛋白、N-钙粘蛋白、β-连环蛋白、c-Myc和CyclinD1的蛋白水平,免疫荧光法检测波形蛋白的表达。通过qPCR检测SFRP1启动子中H3K27me3的富集水平。结果与癌旁组织相比,癌组织中EZH2的mRNA及蛋白水平升高(P<0.05)。细胞活力结果显示,si-EZH2-1组细胞活力较si-NC组有所下降(P<0.05)。Western blot和免疫荧光法检测结果显示,si-EZH2-1组E-钙粘蛋白的表达明显增加,而N-钙粘蛋白的表达明显降低(P<0.05),波形蛋白的荧光强度减弱。与si-NC组相比,si-EZH2-1组的H3K27me3蛋白水平明显降低(P<0.05)。ChIP-qPCR检测结果显示,si-EZH2-1组SFRP1启动子中H3K27me3的富集水平较si-NC组显著降低(P<0.05)。qRT-PCR和Western blot检测结果显示,si-EZH2-1组中SFRP1的表达明显高于si-NC组(P<0.05)。结论EZH2可以促进喉癌细胞的EMT发生,为喉癌临床管理提供了一种全新的策略。
Objective The role of enhancer of zeste homolog 2(EZH2)in epithelial mesenchymal transition(EMT)in laryngeal carcinoma remains unclear.The purpose of this study was to investigate the role of EZH2 in EMT of laryngeal cancer cells.Methods Laryngeal cancer cell lines were divided into:TU177,SNU46,M4E,Tu686 and NP69,human nasopharyngeal epithelial cells(HNPECs).TU177 and Tu686 cell lines with high expression of EZH2 protein were selected to transfect EZH2si RNA.Among them,the group with high transfection effect was EZH21 group,the group with low transfection effect(silencing EZH2)was EZH22 group,and the control group was si NC group transfected with empty vector.The expression of EZH2,secreted frizzled related protein 1(SFRP1)and trimethylation at lysine 27 of histone H3(H3K27me3)in laryngeal carcinoma tissues and cells was detected by qRT PCR and Western blot.After up-regulation of si EZH2,si SFRP1,oe SFRP1 or H3K27me3,the cell viability was detected by CCK-8;the protein levels of E-cadherin,N-cadherin,β-catenin,c-Myc and CyclinD1 were detected by Western blotting,and the expression of vimentin was detected by immunofluorescence.The enrichment level of H3K27me3 in SFRP1 promoter was detected by qPCR.Results Compared with the paracancerous tissues,the mRNA and protein levels of EZH2 in the cancerous tissues were significantly higher(P<0.05).The results of cell viability showed that the cell viability of si EZH21 group was lower than that of si NC group(P<0.05).The results of Western blot and immunofluorescence showed that the expression of E-cadherin was significantly increased,while the expression of N-cadherin decreased significantly(P<0.05),and the fluorescence intensity of vimentin decreased.The level of H3K27me3 protein in si EZH21 group was significantly lower than that in si NC group.The results of ChIP qPCR showed that the enrichment level of H3K27me3 in SFRP1 promoter in si EZH21 group was significantly lower than that in si NC group.The results of qRT PCR and Westernblot showed that the expression of SFRP
作者
王雨洁
张婷
王志颐
季俊峰
吴明海
潘晗
陈伟
WANG Yujie;ZHANG Ting;WANG Zhiyi;JI Junfeng;WU Minghai;PAN Han;CHEN Wei(Nanjing University of Chinese Medicine,Nanjing 210023,Jiangsu,China;Department of Otorhinolaryngology Head and Neck Surgery,General Hospital of Eastern Theater Command,PLA,Nanjing 210002,Jiangsu,China;Jinling Clinical Medical College of Nanjing University of Chinese Medicine/General Hospital of Eastern Theater Command,PLA,Nanjing 210002,Jiangsu,China)
出处
《医学研究与战创伤救治》
CAS
北大核心
2023年第1期20-26,共7页
Journal of Medical Research & Combat Trauma Care
基金
中国博士后科学基金(2018T111165
2017M623435)。
