CTHRC1通过活化FAK-ERK1/2信号通路促进膀胱癌5637细胞迁移和侵袭

CTHRC1 promotes the migration and invasion of bladder cancer 5637 cells through activating the FAK-ERK1/2 signaling pathway

目的:探讨胶原三螺旋重复蛋白1(CTHRC1)在膀胱癌组织和细胞中的表达及其对膀胱癌5637细胞迁移和侵袭的影响及其机制。方法:利用TCGA和Arrayexpress数据库中膀胱癌基因表达数据,分析CTHRC1转录和翻译水平。收集2014年9月至2020年12月重庆医科大学附属第一医院手术切除的144例膀胱癌组织和25例全膀胱切除的癌旁组织标本,以及人膀胱癌细胞RT4、5637、T24、UMUC-3、TCCSUP和输尿管上皮永生化细胞SV-HUC-1。采用免疫组织化学染色法、qPCR法和WB法检测膀胱癌组织和细胞中CTHRC1的表达水平,通过Kaplan-Meier曲线分析CTHRC1表达对总生存期(OS)的影响。运用RNAi技术,敲降5637细胞CTHRC1表达后,通过细胞划痕实验和Transwell实验检测CTHRC1表达下调对5637细胞迁移和侵袭的影响。利用基因集富集分析(GSEA)预测CTHRC1相关的潜在信号通路,WB法检测敲降CTHRC1表达对FAK-ERK1/2通路相关蛋白表达的影响。结果:CTHRC1的转录和翻译水平在肌层浸润性膀胱癌(MIBC)组织和细胞中表达显著上调(均P<0.05),CTHRC1高表达组患者5年OS较低表达患者缩短(P<0.05)。干扰CTHRC1表达后,膀胱癌5637细胞迁移及侵袭能力均显著降低(均P<0.01)。GSEA预测显示,CTHRC1高表达组主要富集在黏着斑激酶(FAK)、肌动蛋白细胞骨架调节、FAK和ERK1/2信号通路。WB法实验结果表明,重组CTHRC1蛋白促进膀胱癌5637细胞FAK-ERK1/2信号通路活化(P<0.05或P<0.01)。结论:CTHRC1在MIBC中表达上调,且与膀胱癌患者不良预后密切相关;CTHRC1促进膀胱癌细胞迁移和侵袭,该过程可能与FAK-ERK1/2信号通路的激活有关。

Objective:To investigate the expression of collagen triple helix repeat containing-1(CTHRC1)in bladder cancer tissues and cells and its effect on the migration and invasion of bladder cancer 5637 cells as well as the related mechanisms.Methods:By referring to the bladder cancer gene expression data in TCGA and Arrayexpress databases,CTHRC1 transcriptional and translational levels were analyzed.144 cases of bladder cancer tissues and 25 cases of para-cancerous tissues resected in the First Affiliated Hospital of Chongqing Medical University from September 2014 to December 2020 were collected for this study;in addition,human bladder cancer cells(RT4,5637,T24,UMUC-3 and TCCSUP)and immortalized human ureteral epithelial SV-HUC-1 cells were also collected.The expression levels of CTHRC1 in human bladder cancer tissues and cell lines were detected by immunohistochemistry staining,qPCR and WB.The Kaplan-Meier curve was used to analyze the effect of CTHRC1 expression on overall survival(OS).CTHRC1expression in 5637 cells was interfered using RNAi technology,and then the effects of CTHRC1 knockdown on migration and invasion of bladder cancer 5637 cells were detected by scratch and Transwell assays.Gene set enrichment analysis(GSEA)was used to predict potential signaling pathways associated with CTHRC1.WB was utilized to detect the effect of CTHRC1 knockdown on the expression of FAK-ERK1/2 pathway related proteins.Results:The transcriptional and translational levels of CTHRC1 were significantly up-regulated in muscle-invasive bladder cancer tissues and cells(all P<0.05),and the 5-year OS was significantly shorter in the patients with high CTHRC1 expression as compared with those with low expression(P<0.05).The migration and invasion of bladder cancer 5637 cells were reduced following CTHRC1 knockdown(all P<0.01).GSEA showed that the CTHRC1 high-expression group was mainly enriched in focal adhesion kinase(FAK),regulation of actin cytoskeleton,and FAK-ERK1/2 signaling pathways.WB assay showed that recombinant CTHRC1 protein promoted the activation of FAK-ERK1/2 signaling pathway in bladder cancer 5637cells(P<0.05 or P<0.01).Conclusion:CTHRC1 expression is up-regulated in invasive bladder cancer and closely related to the poor prognosis of bladder cancer patients.CTHRC1 enhances the invasion and metastasis of bladder cancer cells,which may be associated with the activation of the FAK-ERK1/2 signaling pathway.