Nanog调控细胞周期相关蛋白影响HepG2细胞增殖能力

doi:10.11904/j.issn.1002-3070.2016.05.008

实用肿瘤学杂志 ›› 2016, Vol. 30 ›› Issue (5): 425-429.doi: 10.11904/j.issn.1002-3070.2016.05.008

Nanog调控细胞周期相关蛋白影响HepG2细胞增殖能力

杨晓文¹, 于爱清², 杨毅³

1.湖北省十堰市太和医院检验部(十堰 442000);
2.武汉大学中南医院基因诊断中心;
3.武汉大学第二临床学院

收稿日期:2016-07-03 出版日期:2016-10-28 发布日期:2016-11-02
通讯作者: 杨晓文,E-mail:simpleyxw@sina.com
作者简介:杨晓文,女,(1987-),硕士,检验技师,从事临床检验诊断学相关工作的研究

Nanog influences the proliferative ability of HepG2 cells through the modulation of the cell cycle related proteins

YANG Xiaowen¹, YU Aiqing², YANG Yi³

1.Department of Clinical Laboratory,Taihe Hospital,Shiyan 442000,China;
2.Center of Medical Laboratory,Zhongnan Hospital,The Second Clinical College of Wuhan University;
3.The Second Clinical College of Wuhan University

Received:2016-07-03 Online:2016-10-28 Published:2016-11-02

摘要/Abstract

摘要： 目的研究多能性因子Nanog对HepG2细胞周期相关蛋白表达的影响,进而探索其对HepG2细胞增殖能力的影响。方法本研究通过基因编辑工具TALENs介导Nanog突变而下调其表达,T7E1内切核酸酶和基因测序分析Nanog突变率,Western blot检测Nanog蛋白表达水平,RT-PCR检测Nanog mRNA表达水平,实时细胞活性检测系统检测野生型HepG2细胞和Nanog突变的单克隆HepG2细胞的增殖能力。结果 TALENs成功介导Nanog基因突变,两次转染Nanog-TALENs质粒后,T7E1酶切分析混合细胞的打靶效率近40%。突变的单克隆HepG2细胞Nanog mRNA表达水平较野生型HepG2细胞表达下调3.4倍,而Nanog蛋白表达水平表达下调3.6倍。Nanog突变的单克隆HepG2细胞细胞周期相关蛋白CyclinD1/D3、CyclinE1和CDK2较野生型HepG2细胞表达均下调,Nanog突变的单克隆HepG2细胞表现出明显减弱的增殖能力。结论本研究证实了Nanog通过调控细胞周期相关蛋白CyclinD1/D3、CyclinE1和CDK2影响HepG2细胞增殖,下调Nanog表达可能通过调控细胞周期相关蛋白的表达而抑制HepG2细胞增殖能力。

关键词: TALENs, Nanog, HepG2细胞, 肝癌

Abstract: Objective To study the effect of pluripotency factor Nanog on the expression of the cell cycle related proteins,and then to explore its effect on the proliferative ability of HepG2 cells.Methods TALENs gene editing tool was employed to induce mutation and downregulation expression of Nanog.T7 endonuclease 1 and genomic sequencing was used to analyze the mutation efficiency of Nanog.RT-PCR and western blot were used to determine the expression of mRNA and protein of Nanog,respectively.Real-time cell based assay system was used to measure the proliferative ability of wild-type HepG2 cells and monoclonal HepG2 cells with Nanog mutation.Results TALENs successfully induced mutation of Nanog gene.The targeting efficiency of mixed cells was analyzed by T7 endonuclease 1 approached 40% after two transfection with plasmid of Nanog-TALENs.Additionally,the Nanog mRNA expression level of monoclonal HepG2 with Nanog mutation was downregulated by 3.4 times compared to the wild type HepG2 cells,and the Nanog protein expression level was downregulated by 3.6 times.The cell cycle related proteins CyclinD1/D3,CyclinE1 and CDK2 expression were downregulated in monoclonal HepG2 with Nanog mutation in comparison to the wild type HepG2 cells.Conclusion Nanog plays a role in influencing the proliferative ability of HepG2 cells through modulating the expression of the cell cycle related proteins CyclinD1/D3,CyclinE1 and CDK2.The downregulation expression of Nanog can inhibit the proliferative capacity of HepG2 cells via the regulation of the cell cycle related proteins.

Key words: TALENs, Nanog, HepG2 cells, Hepatocellular carcinoma

中图分类号:

杨晓文, 于爱清, 杨毅. Nanog调控细胞周期相关蛋白影响HepG2细胞增殖能力[J]. 实用肿瘤学杂志, 2016, 30(5): 425-429.

YANG Xiaowen, YU Aiqing, YANG Yi. Nanog influences the proliferative ability of HepG2 cells through the modulation of the cell cycle related proteins[J]. PRACTICAL ONCOLOGY JOURNAL, 2016, 30(5): 425-429.

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Nanog调控细胞周期相关蛋白影响HepG2细胞增殖能力

Nanog influences the proliferative ability of HepG2 cells through the modulation of the cell cycle related proteins

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