HSF1在HBx促进肝细胞癌进展中的作用研究

Investigation of the role of HSF1 in HBx-driven progression of hepatocellular carcinoma

摘要

目的 探讨热休克转录因子1（heat shock transcription factor 1，HSF1）在乙型肝炎病毒X蛋白（hepatitis B virus X protein，HBx）促进肝细胞癌（hepatocellular carcinoma，HCC）细胞迁移、侵袭中的重要作用，并初步研究HSF1介导HBx促进HCC进展的作用机制。 方法 通过4D Label-free定量蛋白质组学和Western blot检测发现HBx对HSF1表达水平的影响；在Huh7和MHCC97H肝癌细胞系中过表达HBx，运用RT-PCR、Western blot检测HBx对HSF1 mRNA、蛋白质水平、HBx蛋白稳定性的影响；运用TCGA数据库分析HSF1在HBV相关性肝癌组织中的表达水平及其与肝癌分期分级、患者预后的关系，并通过Westen blot检测HSF1在HBV相关性肝癌组织中的表达水平；在HCC细胞中过表达HBx并敲减HSF1或用HSF1特异性抑制剂KRIBB11处理细胞，运用Transwell迁移和侵袭实验、划痕实验及F-肌动蛋白染色实验，分析HSF1在HBx促进HCC细胞迁移和侵袭中的作用；运用GEO数据集和HCMDB数据库分析HSF1的下游靶点，并鉴定下游靶点c-Myb在HSF1介导HBx促进HCC进展中的作用。 结果 HBx上调HSF1蛋白质水平而对其mRNA水平无明显影响，HBx通过增强HSF1的蛋白稳定性促进其表达；HSF1在HBV相关性肝癌组织中高表达，HSF1高表达与肝癌分期分级、患者不良预后相关；HBx过表达明显促进Huh7和MHCC97H细胞的迁移、侵袭、划痕愈合能力和伪足形成能力，而HSF1敲减或KRIBB11处理显著减弱HBx促进HCC细胞迁移和侵袭；HSF1促进转移相关蛋白c-Myb的表达，在HSF1敲减的HCC细胞中过表达c-Myb恢复HBx对HCC细胞迁移和侵袭的促进作用。 结论 HBx增强HSF1蛋白稳定性并促进其表达，HSF1上调c-Myb在HBx促进HCC细胞迁移和侵袭中发挥重要作用，靶向HSF1可能有助于减缓HBV相关性HCC进展。

Abstract

Objective To investigate the role of heat shock transcription factor 1（HSF1） in hepatitis B virus X protein（HBx）-driven migration and invasion of hepatocellular carcinoma（HCC） cells，and to preliminarily explore the mechanism of HSF1 mediating HBx-driven HCC progression. Methods 4D label-free quantitative proteomics and Western blot were used to analyze the effect of HBx on HSF1 expression. HBx was overexpressed in the HCC cell lines Huh7 and MHCC97H，and its impact on the mRNA and protein levels and stability of HSF1 was assessed by RT-PCR and Western blot. The Cancer Genome Atlas database was used to analyze the expression of HSF1 in hepatitis B virus（HBV）-associated HCC tissues and its relationship with tumor stage/grade and patient prognosis，and Western blot was used to measure the expression of HSF1 in HBV-associated HCC tissues. HBx was overexpressed in HCC cells，followed by HSF1 knockdown or cell treatment with the HSF1 inhibitor KRIBB11，and Transwell migration and invasion assay，scratch assay，and F-actin staining experiment were performed to analyze the role of HSF1 in HBx-driven HCC cell migration and invasion. GEO and HCMDB datasets were used to identify the downstream target of HSF1，and the role of downstream target c-Myb in HSF1-mediated HBx-driven HCC progression. Results HBx upregulated HSF1 protein levels without significantly affecting its mRNA expression，through enhancing HSF1 protein stability. HSF1 was highly expressed in HBV-associated HCC tissues，and its elevated expression correlated with tumor stage/grade and poor prognosis. HBx overexpression significantly promoted the migration，invasion，wound-healing capacity，and pseudopodia formation capacity of Huh7 and MHCC97H cells，while HSF1 knockdown or KRIBB11 treatment significantly attenuated the HBx-driven migration and invasion of HCC. HSF1 promoted the expression of the metastasis-associated protein c-Myb，and c-Myb overexpression in HSF1-knockdown HCC cells restored the promotive effect of HBx on HCC cell migration and invasion. Conclusion HBx enhances HSF1 protein stability to promote its expression. Upregulation of c-Myb by HSF1 plays a pivotal role in HBx-driven HCC cell migration and invasion. Targeting HSF1 may help to delay the progression of HBV-associated HCC.

关键词

热休克转录因子1 / 乙型肝炎病毒X蛋白 / 肝细胞癌 / 迁移 / 侵袭

Key words

heat shock transcription factor 1 （HSF1） / HBx / hepatocellular carcinoma （HCC） / migration / invasion

基金资助

重庆市自然科学基金面上资助项目(CSTB2022NSCQ-MSX0864)