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蛋白精氨酸N-甲基转移酶4上调八聚体结合转录因子4表达促进胃癌细胞的干性
Protein arginine N-methyltransferase 4 up-regulates Oct4 expression and promotes stemness of gastric cancer cells
- 第三军医大学学报 2018年40卷第8期 页码：679-685
- 作者机构：
1.400715 重庆，西南大学生命科学学院
2.400037 重庆，陆军军医大学(第三军医大学)第二附属医院消化内科
3.402160 重庆，重庆医科大学附属永川医院药学部
- 作者简介：
[杨仕明，E-mail:shimingyang@yahoo.comYANG Shiming, E-mail:shimingyang@yahoo.com
[杨应斌，E-mail:yybin@swu.edu.cnYANG Yingbin, E-mail:yybin@swu.edu.cn
- 基金信息：国家自然科学基金面上项目(81572460)
DOI：10.16016/j.1000-5404.201711211
中图分类号： R345.5;R394.3;R735.2收稿：2017-11-24，
修回：2018-01-18，
纸质出版：2018-04-30
- 稿件说明：
移动端阅览
吴玉云, 杨泓莹, 覃勇, 等. 蛋白精氨酸N-甲基转移酶4上调八聚体结合转录因子4表达促进胃癌细胞的干性[J]. 第三军医大学学报, 2018,40(8):679-685.
Yuyun WU, Hongying YANG, Yong QIN, et al. Protein arginine N-methyltransferase 4 up-regulates Oct4 expression and promotes stemness of gastric cancer cells[J]. Journal of Third Military Medical University, 2018, 40(8): 679-685.
吴玉云, 杨泓莹, 覃勇, 等. 蛋白精氨酸N-甲基转移酶4上调八聚体结合转录因子4表达促进胃癌细胞的干性[J]. 第三军医大学学报, 2018,40(8):679-685. DOI： 10.16016/j.1000-5404.201711211.
Yuyun WU, Hongying YANG, Yong QIN, et al. Protein arginine N-methyltransferase 4 up-regulates Oct4 expression and promotes stemness of gastric cancer cells[J]. Journal of Third Military Medical University, 2018, 40(8): 679-685. DOI： 10.16016/j.1000-5404.201711211.
摘要
目的
2
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
2
以pcDNA3.1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
ChIP)实验证实PRMT4与Oct4基因启动子结合情况。
结果
2
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
2
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。
Abstract
Objective
2
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
2
A eukaryotic plas
mid 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 (ChIP) was employed to demonstrate the enrichment of PRMT4 in Oct4 gene promoter.
Results
2
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 ChIP confirmed the binding of PRMT4 to Oct4 promoter region.
Conclusion
2
PRMT4 interacts with the promoter region of Oct4 gene to enhance Oct4 expression
thereby promoting the stem-like traits of gastric cancer cells.
关键词
Keywords
references
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