变异型EBER2通过抑制PKR通路增强鼻咽癌细胞抗凋亡能力

doi:10.3969/j.issn.1004-616x.2018.05.001

癌变·畸变·突变 ›› 2018, Vol. 30 ›› Issue (5): 333-338,344.doi: 10.3969/j.issn.1004-616x.2018.05.001

• 论著 • 下一篇

变异型EBER2通过抑制PKR通路增强鼻咽癌细胞抗凋亡能力

贺荟静¹, 刘金成¹, 刘芡芡², 王云¹

1. 青岛大学医学院病原生物学系, 山东青岛 266021;
2. 山东省莱钢医院检验科, 山东莱芜 271126

收稿日期:2018-05-01 修回日期:2018-09-06 出版日期:2018-09-30 发布日期:2018-09-30
通讯作者: 王云,E-mail:qdwangyun@aliyun.com E-mail:qdwangyun@aliyun.com
作者简介:贺荟静,E-mail:1071559089@qq.com。
基金资助:
国家自然科学基金项目（81171571）

A variant of Epstein-Barr virusencoded small RNA 2 enhanced resistance to apoptosis by inhibiting the PKR pathway in nasopharyngeal carcinoma cell lines

HE Huijing¹, LIU Jincheng¹, LIU Qianqian², WANG Yun¹

1. School of Pathogenic Biology, Qingdao University, Qingdao 266021;
2. Department of Clinical Laboratory, Laigang Hospital, Laiwu 271126, Shandong, China

Received:2018-05-01 Revised:2018-09-06 Online:2018-09-30 Published:2018-09-30

摘要/Abstract

摘要： 目的：前期研究发现变异型EB病毒编码小RNA 2（EBER2）基因EB-8m可能与鼻咽癌（NPC）相关，本研究旨在探讨变异型EBER2是否通过增强对RNA激活蛋白激酶（PKR）的抑制作用从而提高NPC细胞的抗凋亡能力。方法：以野生型和EB-8m变异型EBER2基因稳定转染的EB病毒阴性NPC细胞系HONE1和CNE1为研究对象，以未转染EBER2基因细胞为对照，分别用10 000 IU/mLα-干扰素（IFN-α）和2μg/mL顺铂诱导凋亡后以流式细胞术检测细胞凋亡；采用Western blot检测IFN-α诱导凋亡细胞中PKR及下游eIF2α和c-Jun的磷酸化蛋白及Bcl-2蛋白表达水平。对EBER2基因转染细胞进行RNA和PKR蛋白免疫共沉淀，检测共沉淀产物中EBER2富集倍数。结果：EBER2基因转染细胞的凋亡率低于未转染EBER2基因细胞（P < 0.05或P < 0.01），且变异型的细胞凋亡率低于野生型（P < 0.05）。与未转染EBER2基因细胞比较，EBER2转染细胞中磷酸化PKR、eIF2α和c-Jun减少，Bcl-2表达升高，且磷酸化PKR在变异型转染HONE1和CNE1细胞中的水平低于野生型转染细胞，磷酸化eIF2α在变异型转染HONE1细胞中的水平亦低于野生型转染细胞，差异均有统计学意义（P均 < 0.05）。EBER2基因转染细胞RNA和PKR免疫共沉淀产物中可检测到EBER2，且变异型的富集倍数高于野生型（P < 0.05）。结论：EB-8m变异型EBER2可能增强EBER2对PKR的结合作用，同时抑制eIF2α和c-Jun磷酸化并上调Bcl-2表达，进而增强NPC细胞抗凋亡能力。

关键词: EB病毒, EBER2, 鼻咽癌, 基因变异, RNA激活蛋白激酶

Abstract: OBJECTIVE:Previous studies demonstrated that the EB-8m variant of Epstein-Barr virus (EBV)-encoded small RNA 2 (EBER2) might be associated with nasopharyngeal carcinoma (NPC). The aim of this study was to investigate whether the variant EBER2 would confer resistance to apoptotic by enhancing inhibition of RNA-activated protein kinase (PKR) in NPC cell lines. METHODS:The cell anti-apoptosis observation induced by 10 000 IU/mL interferon-α(IFN-α) or 2μg/mL cisplatin was conducted in EBV-negative NPC cell lines (CNE1 and HONE1) with stable expression of wild type or EB-8m variant EBER2 gene under the control of lentivirus vector transfection group. Western blotting was performed to determine phosphorylation of PKR, eIF2α and c-Jun (which are down streams of PKR activation),expression of Bcl-2 in IFN-α-treated cells with stable expression of EBER2 and control cells. RNA-binding protein immunoprecipitation against PKR was assayed in cells with stable expression of EBER2 to detect the fold enrichment of EBER2 with PKR. RESULTS:EBER2 -expressing NPC cells enhanced anti-apoptosis ability in contrast to the control cells (P < 0.05 or P < 0.01),and the cells expressing variant EBER2 showed higher anti-apoptosis ability compared with those expressing wild type EBER2 (P < 0.05). Compared with the control cells,EBER2-expressing cells showed lower level of phosphorylation of PKR, eIF2α and c-Jun and elevation of Bcl-2 (P < 0.05). The levels of phosphorylated PKR in variant EBER2 transfected HONE1 and CNE1 cells were significantly lower than that in wild type EBER2 transfected cells (P < 0.05). That of phosphorylated eIF2α in variant EBER2 transfected HONE1 cells was also significantly lower than that in wild type EBER2 transfected cells (P < 0.05). EBER2 could be detected in immunoprecipitation against PKR, and the variant EBER2 had higher fold enrichment than wild type EBER2 (P < 0.05). CONCLUSION:The EB-8m variant EBER2 demonstrated its inhibition of PKR,and of phosphorylation of eIF2α and c-Jun,and up-regulated Bcl-2 expression,thus enhanced the anti-apoptosis ability of NPC cell lines.

Key words: Epstein-Barr virus, EBER2, nasopharyngeal carcinoma, gene variation, RNA-activated protein kinase

中图分类号:

R730.2

贺荟静, 刘金成, 刘芡芡, 王云. 变异型EBER2通过抑制PKR通路增强鼻咽癌细胞抗凋亡能力[J]. 癌变·畸变·突变, 2018, 30(5): 333-338,344.

HE Huijing, LIU Jincheng, LIU Qianqian, WANG Yun. A variant of Epstein-Barr virusencoded small RNA 2 enhanced resistance to apoptosis by inhibiting the PKR pathway in nasopharyngeal carcinoma cell lines[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(5): 333-338,344.

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变异型EBER2通过抑制PKR通路增强鼻咽癌细胞抗凋亡能力

A variant of Epstein-Barr virusencoded small RNA 2 enhanced resistance to apoptosis by inhibiting the PKR pathway in nasopharyngeal carcinoma cell lines

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