Hsp60表达下调通过促进TGFBI分泌增强膀胱癌细胞侵袭转移

Down-regulated Hsp60 expression enhances invasion and metastasis of bladder cancer cells by promoting TGFBI secretion

目的探索Hsp60蛋白表达对膀胱癌细胞侵袭转移的影响。方法免疫组化检测膀胱癌组织芯片Hsp60蛋白的表达,统计学分析其与膀胱癌患者临床参数的相关性;利用慢病毒载体构建Hsp60稳定干涉的膀胱癌细胞株,Transwell及划痕实验检测Hsp60表达对膀胱癌细胞侵袭转移的影响。RNA-seq分析Hsp60稳定干涉后基因表达的差异,筛选出与侵袭转移相关的基因。Western-bolt及ELISA验证差异基因在蛋白水平的变化,功能学验证差异基因在Hsp60诱导的膀胱癌侵袭转移中的作用,并探索干涉Hsp60表达影响差异基因表达的机制。结果Hsp60蛋白特异性表达在膀胱癌细胞的胞质中,且在浸润性膀胱癌组织中的表达显著降低。统计学结果显示:Hsp60的表达与膀胱癌的转移、临床分级及复发显著相关;细胞学实验证实:干涉Hsp60表达后膀胱癌细胞的侵袭迁移能力显著增强的同时TGFBI的表达及分泌显著升高;利用siRNA阻断Hsp60稳定干涉膀胱癌细胞的TGFBI表达后,可显著抑制Hsp60干涉所造成的膀胱癌细胞侵袭迁移增强。进一步研究发现:Hsp60干涉后膀胱癌细胞线粒体ROS水平及HIF1α的表达均显著升高。利用MitoEMPO降低ROS水平或者利用siRNA阻断HIF1α表达后,Hsp60干涉引起的TGFBI表达及分泌升高可被显著抑制。结论Hsp60表达降低通过促进TGFBI表达增强膀胱癌细胞的侵袭转移,Hsp60表达降低通过ROS/HIF1α信号通路促进TGFBI的表达。

Objective To investigate the effects of Hsp60 down-expression on the invasion and metastasis of bladder cancer cells.Methods The protein expression of Hsp60 in bladder cancer was determined with immunohistochemistry.The correlation between Hsp60 protein expression and pathological parameters of bladder cancer was analyzed.Bladder cancer cell line with stable interference of Hsp60 was construected with shRNA lentivirus vectors.The effects of Hsp60 expression on bladder cancer cell migration and invasion were detected with transwell assay and wound healing assay.The gene expression differneces after Hsp60 knockdown were analyzed with RNA-seq,and metastasis-related genes were screened.The protein expressions of metastasis-related genes were determined with Western-bolt and ELISA.The role of differential genes in Hsp60-induced invasion and metastasis of bladder cancer was cerified with functional studies,and the specific mechanism of Hsp60 expression affecting differential genes expression was explored.Results Hsp60 was specifically expressed in the cytoplasm of bladder cancer cells and was significantly reduced in invasive bladder cancer tissues.The expression of Hsp60 was significantly correlated with metastasis,clinical grade and recurrence of bladder cancer.Hsp60 knockdown significantly promoted the invasion and metastasis of bladder cancer cells and significantly improved the expression and secretion of TGFBI.Knock down of TGFBI expression by siRNA in bladder cancer cells with Hsp60-knockdown significantly inhibited the metastasis and invision of bladder cancer.The levels mitochondrial ROS and HIF1a were significantly increased in bladder cancer cells with Hsp60 knockdown.After MitoEMPO was used to reduce ROS level or siRNA was used to block HIF1a expression,the increased expression and secretion of TGFBI induced by Hsp60 knockdown was significantly inhibited.Conclusion Decreased Hsp60 expression enhances the invasion and metastasis of bladder cancer cells by inducing TGFBI expression through the ROS/HIF1a pathway.