HECW2通过被CREB1转录激活而增强KIRC细胞活力并促进细胞侵袭和迁移  

HECW2 enhances viability,invasion and migration of KIRC cells under transcriptional activation by CREB1

作　　者：沈慧 寇茜睿 王丽[1] 张静[1] 李芳 SHEN Hui;KOU Qianrui;WANG Li;ZHANG Jing;LI Fang(Medical School of Yan'an University,Yan'an 716000,China)

机构地区：[1]延安大学医学院,陕西延安716000

出　　处：《中国病理生理杂志》2023年第7期1163-1173,共11页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82260530);陕西省自然科学基础研究计划资助项目(No.2022JQ-907);陕西省高校科协青年人才托举计划项目(No.20210309);延安大学博士科研启动项目(No.YDBK2020-04)。

摘　　要：目的:探究E3泛素连接酶HECW2(HECT,C2 and WW domain containing E3 ubiquitin protein ligase 2)在肾透明细胞癌(kidney renal clear cell carcinoma,KIRC)细胞活力、凋亡、侵袭和迁移中的作用及其分子调控机制。方法:借助GEPIA(Gene Expression Profiling Interactive Analysis)数据库分析HECW2在泛癌中的表达水平,同时利用SangerBox数据库分析HECW2表达水平与泛癌免疫浸润和预后的关系。在此基础上,进一步通过体外实验探究HECW2在KIRC中的调控作用及相关分子机制。首先,通过RT-qPCR和Western blot分析HECW2在KIRC细胞系中的表达水平;其次,通过CCK-8实验分析敲减HECW2对KIRC细胞活力的影响;再者,通过流式细胞术分析敲减HECW2对KIRC细胞凋亡的影响;最后,通过划痕实验和Transwell实验分析敲减HECW2对KIRC细胞迁移和侵袭的影响。此外,通过Western blot和ChIP-qPCR探究HECW2是否在转录因子cAMP反应元件结合蛋白1(cAMP response element-binding protein 1,CREB1)的转录激活作用下影响KIRC细胞。结果:泛癌分析结果显示,HECW2在KIRC和胰腺癌中显著高表达,而在肺腺癌和肺鳞状细胞癌中显著低表达,且HECW2表达水平与这4种肿瘤的免疫浸润水平呈显著正相关;但HECW2高表达组和低表达组相比,仅KIRC患者生存期长短有显著差异,即HECW2表达水平越高,KIRC患者总生存期、疾病特异性生存期和无进展间期越长,预后越好。体外实验结果表明,HECW2在KIRC组织和细胞系中均呈显著高表达;敲减HECW2可显著抑制KIRC细胞活力、侵袭和迁移,并促进其凋亡。敲减CREB1可显著降低KIRC细胞中HECW2的mRNA和蛋白表达水平,而过表达CREB1则观察到相反的结果。ChIP-qPCR实验结果表明CREB1能够与HECW2的启动子区域结合,提示HECW2能够被CREB1转录激活。结论:HECW2可在CREB1的转录激活作用下增强KIRC细胞活力并促进细胞侵袭和迁移。AIM:To investigate the role of HECT,C2 and WW domain containing E3 ubiquitin protein ligase 2(HECW2)in the viability,apoptosis,migration and invasion of kidney renal clear cell carcinoma(KIRC)cells and the underlying molecular mechanism.METHODS:The expression levels of HECW2 in pan-cancer were analyzed by Gene Expression Profiling Interactive Analysis(GEPIA)database,and SangerBox database was used to determine the relationship between HECW2 expression levels and immune infiltration/prognosis in pan-cancer.Furthermore,the role of HECW2 in KIRC and the underlying mechanisms were explored in vitro experiments.The expression levels of HECW2 in KIRC cell lines were analyzed by RT-qPCR and Western blot.The effect of HECW2 knockdown on the viability of KIRC cells was assessed by CCK-8 assay.The effect of HECW2 knockdown on the apoptosis of KIRC cells was determined by flow cytometry.Wound-healing assay and Transwell assay were used to evaluate the effect of HECW2 knockdown on the migration and invasion of KIRC cells.In addition,Western blot and ChIP-qPCR were used to investigate whether HECW2 affected KIRC cells under the transcriptional activation by cAMP response element-binding protein 1(CREB1).RESULTS:Pan-cancer analysis showed that HECW2 was significantly overexpressed in KIRC and pancreatic adenocarcinoma,but was remarkedly underexpressed in lung adenocarcinoma and lung squamous cell carcinoma,and the expression level of HECW2 was significantly positively correlated with the immune infiltration level of these four cancers above.However,only the survival of KIRC patients had a significant difference between high and low HECW2 expression groups.The higher expression level of HECW2,the longer survival time(such as overall survival,disease-specific survival and progression-free interval)and the better prognosis of KIRC patients.In vitro experiments results showed that HECW2 was significantly highly expressed both in KIRC tissues and cell lines.Knockdown of HECW2 significantly inhibited the viability,invasion and migra

关 键 词：肾透明细胞癌 HECW2蛋白 cAMP反应元件结合蛋白1 细胞活力 细胞侵袭 细胞迁移 

分 类 号：R737.11[医药卫生—肿瘤] R363.2[医药卫生—临床医学]