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热休克蛋白90对人肝癌细胞自噬相关转录因子EB的调控机制

汪梦楠, 刘利霞, 邓耀棠, 陈雪梅^*

南方医科大学公共卫生学院职业卫生与职业医学系，广州 510515

摘要

本文旨在研究热休克蛋白90 (heat shock protein 90, Hsp90)对肝癌细胞自噬相关转录因子EB (transcription factor EB, TFEB)的调控机制。体外培养人肝癌细胞株HepG2，分别用Hsp90的N端抑制剂STA9090和C端抑制剂Novobiocin处理，用 Western blot和RT-PCR检测TFEB和自噬相关蛋白的表达情况，用染色质免疫共沉淀(chromatin immunoprecipitation, ChIP)实验 检测Hsp90与TFEB近启动子区的结合情况，用双荧光素酶报告基因实验检测Hsp90对TFEB启动子活性的影响。结果显示， 低氧诱导HepG2细胞TFEB蛋白和mRNA表达水平上调。在常氧及低氧条件下，用STA9090和Novobiocin分别处理HepG2细胞 后，TFEB和LC3、P62蛋白表达水平均显著下降。转染Hsp90α表达质粒可上调野生型和Hsp90α敲除的HepG2细胞TFEB蛋白 表达水平。Hsp90可结合于TFEB近启动子区，参与调控其转录过程，而STA9090和Novobiocin均可抑制Hsp90与TFEB近启动 子区的结合能力，降低TFEB启动子活性。以上结果提示，Hsp90通过与人肝癌细胞TFEB近启动子区的结合促进TFEB转录， 从而上调自噬相关蛋白LC3、P62表达水平。

关键词： 热休克蛋白90; 转录因子EB; 自噬; 原发性肝癌

分类号：R735.7

Regulatory mechanism of heat shock protein 90 on autophagy-related transcription factor EB in human hepatocellular carcinoma cells

WANG Meng-Nan, LIU Li-Xia, DENG Yao-Tang, CHEN Xue-Mei^*

Department of Occupational Health and Medicine, School of Public Health, Southern Medical University, Guangzhou 510515, China

Abstract

This study was aimed to investigate the regulatory mechanism of heat shock protein 90 (Hsp90) on transcription factor EB (TFEB) during autophagy in liver cancer cells. Human hepatocellular carcinoma cell line HepG2 was treated with Hsp90 N- and C-terminal inhibitors (STA9090 and Novobiocin), respectively. Western blot and RT-PCR were used to detect the expression levels of TFEB and autophagy-related proteins. Chromatin immunoprecipitation (ChIP) assay was used to observe the ability of Hsp90α binding to the TFEB proximal promoter region. The double-luciferase gene reporter experiment was used to determine the activity of TFEB promoter. The results showed that hypoxia induced up-regulation of TFEB protein and mRNA expression levels in the HepG2 cells. The protein expression levels of TFEB, LC3 and P62 were down-regulated significantly by either STA9090 or Novobiocin, under both normoxic and hypoxic conditions. Transfection of Hsp90α-overexpressing plasmids up-regulated TFEB protein levels in either wild-type or Hsp90α knockout HepG2 cells. Hsp90 bound to the TFEB proximal promoter region and was involved in regulating TFEB transcriptional process. Whereas both STA9090 and Novobiocin inhibited Hsp90 to bind to the TFEB proximal promoter region, and decreased the activity of TFEB promoter. These results suggest that Hsp90 promotes TFEB transcription in human hepatocellular carcinoma cells by binding to the proximal promoter region, thereby up-regulating the expression levels of autophagy-related proteins.

Key words: heat shock protein 90; transcription factor EB; autophagy; hepatocellular carcinoma

收稿日期：2019-08-25　　录用日期：2020-02-13

通讯作者：陈雪梅　　E-mail: cxmcsz@smu.edu.cn

DOI: 10.13294/j.aps.2020.0028

引用本文：

汪梦楠, 刘利霞, 邓耀棠, 陈雪梅. 热休克蛋白90对人肝癌细胞自噬相关转录因子EB的调控机制[J]. 生理学报 2020; 72 (2): 157-166.

WANG Meng-Nan, LIU Li-Xia, DENG Yao-Tang, CHEN Xue-Mei. Regulatory mechanism of heat shock protein 90 on autophagy-related transcription factor EB in human hepatocellular carcinoma cells. Acta Physiol Sin 2020; 72 (2): 157-166 (in Chinese with English abstract).