摘要
目的探讨蛋白精氨酸N-甲基转移酶(protein arginine N-methyltransferase,PRMTs)调控八聚体结合转录因子4(octamer-binding transcription factor4,Oct4)的表达及其对胃癌细胞"干性"的影响。方法以pc DNA3.1载体构建PRMT4过表达质粒,转染至人胃腺癌MKN45细胞;倒置相差显微镜下观察肿瘤细胞成球能力的变化;qRT-PCR和Western blot检测胃癌干细胞标志物CD44、Lgr5及"干性"维持分子Nanog、Oct4的改变;继而构建含Oct4基因启动子荧光素酶报告载体,分析PRMT4对Oct4启动子活性的影响;最后采用染色质免疫沉淀(chromatin immunoprecipitation,Ch IP)实验证实PRMT4与Oct4基因启动子结合情况。结果 MKN45细胞转染蛋白精氨酸N-甲基转移酶-4基因过表达载体后,形成肿瘤干细胞球的能力显著增强;随后qRT-PCR及Western blot检测结果显示胃癌干细胞标志物CD44、Lgr5的mRNA及蛋白水平上调;此外,过表达PRMT4可明显增加"干性"维持分子Oct4的mRNA及蛋白水平,而对Nanog则无明显影响;双荧光素酶实验结果显示,与对照组相比,PRMT4过表达组Oct4的启动子活性明显增强(P<0.05);染色质免疫共沉淀实验结果证实,PRMT4可与Oct4启动子结合。结论 PRMT4可结合至Oct4基因启动子上,通过增加启动子活性,从而促进Oct4转录及表达,进而增强胃癌细胞的干性。
Objective To investigate the effect of protein arginine N-methyltransferase 4（ PRMT4）on the expression of octamer-binding transcription factor 4（ Oct4） and on the stemness of gastric cancer cells.Methods A eukaryotic plasmid expressing PRMT4 was constructed and transfected into human gastric cancer cell line MKN45,and the in vitro tumor sphere formation was observed. qRT-PCR and Western blotting were performed to examine the expressions of gastric cancer stem cell surface markers including CD44,Lgr5,Nanog and Oct4 in the transfected cells. Dual luciferase reporter assay was used to test the activity of the Oct4 promoter,and chromatin immunoprecipitation（ Ch IP） was employed to demonstrate the enrichment of PRMT4 in Oct4 gene promoter. Results MKN45 cells transfected with the PRMT4 plasmid showed enhanced ability of tumor sphere formation. qRT-PCR and Western blotting showed that the expression levels of CD44 and Lgr5 were significantly up-regulated in the cells after PRMT4 over-expression,which also resulted in increased expression of Oct4 at both mRNA and protein levels without obviously affecting Nanog expression. Dual luciferase reporter assay demonstrated that PRMT4 enhanced the activity of Oct4 promoter,and the results of Ch IP confirmed the binding of PRMT4 to Oct4 promoter region. Conclusion PRMT4 interacts with the promoter region of Oct4 gene to enhance Oct4 expression,thereby promoting the stem-like traits of gastric cancer cells.
作者
吴玉云
杨泓莹
覃勇
汪苏敏
张亚丽
刘胜伟
杨仕明
杨应斌
WU Yuyun;YANG Hongying;QIN Yong;WANG Sumin;ZHANG Yali;LIU Shengwei;YANG Shiming;YANG Yingbin(College of Life Sciences, Southwest University, Chongqing, 400715;Department of Gastroenterology, Second Affiliated Hospital, Army Military Medical University（Third Military Medical University）, Chongqing, 400037;Department of Pharmacy, Yongchuan Hospital Affiliated to Chongqing Medical University, Chongqing, 402160, China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2018年第8期679-685,共7页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81572460)~~
关键词
蛋白精氨酸N-甲基转移酶
八聚体结合转录因子4
肿瘤干细胞
转录调控
protein arginine N-methyhransferase 4
octamer-binding transcription factor 4
cancerstem cells
transcriptional regulation
