宫颈癌细胞株Hela中CSN6对E6-AP及p53的调节及其分子机制

目的:探讨宫颈癌细胞株Hela中组成型光形态发生因子9信号复合体6(CSN6)对E6-AP及其目标蛋白p53的调节及其分子机制。方法:重组慢病毒干扰载体质粒转染Hela细胞,降调CSN6表达。Western blot法检测E6-AP表达,实时荧光定量PCR检测p53下游基因表达变化。分别将蛋白酶抑制剂MG132、真核生物蛋白合成抑制剂放线菌酮(CHX)和高表达CSN6的Flag-CSN6质粒分别作用于Hela细胞,Western blot法检测E6-AP蛋白表达。将重组慢病毒质粒shRAN CSN6转染Hela细胞48h后,泛素化检测分析CSN6调节E6-AP蛋白表达的分子机制。试验组裸鼠皮下接种shRNA-CSN6细胞株,对照组接种shRNA-vector细胞株,观察肿瘤体积变化。结果:放线菌酮(CHX)可下调E6AP表达,抑制CSN6表达后E6-AP下调更显著;MG132可使E6AP表达增加,CSN6可加强该作用;抑制CSN6表达后,Hela细胞和裸鼠肿瘤组织中p53下游相关基因表达增加,裸鼠肿瘤生长缓慢。泛素化检测提示,CSN6可抑制E6-AP蛋白的泛素化水解,并呈剂量依赖性。结论:宫颈癌细胞株Hela中CSN6上调E6AP是通过抑制E6-AP蛋白的泛素化水解,稳定其在细胞中的表达,实现对p53的持续降解。

The role of CSN6 in regulating E6AP and p53 in cervical cancer

Objective:To explore the molecular mechanism of constitutive photomor-phogenesis 9 subunit 6 ( CSN6 ) on regulating E6 AP and p53 in cervical cancer cell line Hela cells. Methods:The expression of CSN6 was down-regulated by lentivirus-mediated in Hela cells. The expression of E6-AP was detected by Western blot. The expression of p53 downstream genes was detected by real-time PCR. The molecular mechanism of CSN6 regulated the protein expression of E6-AP was detected by transformations and ubiquitination experiments. Results:CSN6 decreased the turnover rate of E6 AP in the presence of the eukaryotic protein synthesis inhibitor cycloheximide(CHX). CSN6-mediated E6AP upregulation was enhanced by the pro-teasome inhibitor MG132 . Overexpression of CSN6 decreased the endogenous ubiquitination lev-el of E6AP in a dose-dependent manner. CSN6 knockdown caused an upregulation of p53 target gene expression,including GADD45,p21,Bax,etc,and reduced the growth of tumor in nude mice. Conclusion:CSN6 is a critical ubiquitination regulator of E6AP and can positively affect the steady-state expression of E6AP.