乙型肝炎病毒大表面蛋白诱导内质网应激调控p27 IRES翻译活性的机制研究
Regulation mechanism of p27 IRES translation activity under large HBV surface protein-induced endoplasmic reticulum stress
郭亦笑 1邵杰 1尉丁 1陈志南 1边惠洁1
作者信息
- 1. 国家分子医学转化中心,空军军医大学基础医学院细胞生物学教研室,陕西西安 710032
- 折叠
摘要
目的 探讨在乙型肝炎病毒大表面蛋白(LHB)诱导的内质网应激条件下,抑癌基因p27 5'UTR内部核糖体进入位点(IRES)的活性变化及其调控机制.方法 构建双荧光素酶报告质粒,用双荧光素酶报告基因实验检测p27 5'UTR中IRES的活性;利用qRT-PCR和Western blotting检测LHB诱导的未折叠蛋白反应相关分子水平;RNA下拉、质谱技术和RNA免疫沉淀筛选并确认潜在的与p27 5'UTR结合的相关蛋白;siRNA转染建立PCBP2和ANXA2敲低细胞模型,用双荧光素酶报告基因检测在敲低PCBP2和ANXA2后p27 IRES翻译活性的变化.结果 成功构建了检测p27 IRES活性的双荧光素酶报告质粒.LHB可以诱导内质网应激,同时显著提高p27的IRES翻译活性(P＜0.01).LHB过表达激活了 PERK通路并提高了 eIF2α的磷酸化水平,特异性PERK通路抑制剂GSK2606414使RERK和eIF2α的磷酸化水平降低,从而引起p27 5'UTR的IRES翻译活性显著降低(P＜0.01,P＜0.05).同时,磷酸酶抑制剂Sa1003能显著增加eIF2α磷酸化水平,使p27 IRES活性进一步增强(P＜0.01,P＜0.05).最后,我们发现PCBP2和ANXA2作为潜在的反式作用因子可增强p27 IRES活性.结论 LHB诱导的内质网应激通过eIF2α磷酸化增强p27 IRES翻译活性.反式作用因子PCBP2和ANXA2正向调控其IRES翻译活性.
Abstract
Objective To investigate the activity changes of internal ribosome entry site(IRES)in tumor suppressor gene p27 5'UTR and its regulatory mechanism under endoplasmic reticulum stress induced by large HBV surface protein(LHB).Methods Dual-luciferase reporter plasmid was constructed,and dual-luciferase reporter assay was used to detect the activity of IRES in p27 5'UTR.qRT-PCR and Western blotting were used to detect the expression levels of unfolded protein response-related molecules induced by LHB.RNA pull-down,mass spectrometry and immunoprecipitation were used to screen and verify the potential proteins that bound to p27 5'UTR.The knock-down cell models of PCBP2 and ANXA2 were established with siRNA transfection,and changes in the translation activity of p27 IRES in PCBP2 and ANXA2 knock-down cells were detected by dual-luciferase reporter assay.Results A dual-luciferase reporter plasmid was constructed to detect the activity of p27 IRES.LHB could induce endoplasmic reticulum stress and significantly increased the translation activity of p27 IRES(P＜0.01).Overexpression of LHB activated PERK pathway and increased the phosphorylation of eIF2α.The phosphorylation level of RERK and eIF2α was decreased by using the specific PERK pathway inhibitor GSK2606414,and the IRES translation activity of p27 5'UTR was significantly decreased(P＜0.01,P＜0.05).Meanwhile,phosphatase inhibitor Sal003 significantly increased the phosphorylation level of eIF2α,which further enhanced the activity of p27 IRES(P＜0.01,P＜0.05).Finally,we found that PCBP2 and ANXA2 as potential IRES trans-acting factors,played a positive role in enhancing the activity of p27 IRES.Conclusion LHB-induced endoplasmic reticulum stress enhances the translation activity of p27 IRES through eIF2α phosphorylation.IRES trans-acting factors PCBP2 and ANXA2 positively regulate the translation activity of p27 IRES.
关键词
乙型肝炎病毒大表面蛋白/p27/5'UTR/内质网应激/内部核糖体进入位点/反式作用因子Key words
large HBV surface protein/p27 5'UTR/endoplasmic reticulum stress/internal ribosome entry sites/IRES trans-acting factor引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据