CO-029介导炎症因子TNFα/TNFαR1诱导上皮间变调控胆管癌侵袭转移的研究

Recently, the protumorigenic effects of inflammatory microenviroment in promoting invasion/metastasis has been widely recognoized.Our previous study demonstrated that the overexpression of tetraspanin CO-029 in intrahepatic cholangiocarcinoma(ICC) contributed to tumor invasion and metastasis, and co-immunoprecipitation and mass spectrometry assays revealed CO-029 could complex with inflammatory cytokine TNFα receptor- - TNFαR1. Further study showed that the epithelial-mesenchymal transition(EMT) induced by TNFα in an ICC cell line- - -HCCC-9810 was blocked through knockdown of CO-029 expression with RNA interference.The results obtained thus far led us to investigate whether CO-029 might mediate the inflammatory cytokine TNFα-induced EMT to regulate invasion and metastasis in ICC. To probe this, we herein first employ tissue microarrays(TMAs) to detect the CO-029 expression in large sample of ICC tissues, accompanied with in vitro and in vivo studies,to determine whether the CO-029 expression in ICC to be associated with tumor invasion /metastasis or prognosis. Then, we will examine structurally and functionally whether tetraspanin CO-029 could act on TNFα/TNFαR1 system by assays of RNA interference, laser confocal microscopy, co-immunoprecipitation, Western blot, etc. We further employ vsh-RNA interference to selectively knockdown the expression of CO-029 and/or TNFαR1 in ICC cell lines to determine potential functional mechanism of CO-029 in TNFα/TNFαR1 system. Lastly, we will verify those significance above in nude mice EMT model. Thus, by interpreting the association between CO-029 and TNFα/TNFαR1, and their potential role in EMT and tumor invasion/metastasis, we, for the first time to our knowledge, investigate the potential role of CO-029 as a predictor for tumor invasion/metastasis(and/or as prognostic marker) in ICC, and further, we probe the signal pathways involved in CO-029's vital role in inflammation-induced EMT to regulate ICC invasion/metastasis.

炎性微环境诱导并促进肿瘤侵袭转移已受到极大关注。我们前期研究发现四跨膜蛋白CO-029表达可促进胆管癌侵袭转移，免疫共沉淀联合质谱分析发现炎性因子TNFα之受体-TNFαR1可与CO-029形成复合物，干扰CO-029后TNFα诱导的胆管癌上皮间变被阻断。据此，我们推测并探讨CO-029可能介导TNFα诱导的上皮间变而调控胆管癌侵袭转移。本研究应用组织芯片对大样本肝内胆管癌组织检测并结合体内外实验，明确CO-029表达与胆管癌侵袭转移、复发和预后的关系；激光共聚焦、RNA干扰等研究并结构上明确C0-029与TNFαR1的相互作用；慢病毒干扰CO-029和（或）TNFαR1表达，功能上探讨CO-029对TNFα/TNFαR1的影响及作用机制；最后体内实验证实。本研究首次探讨CO-029作为胆管癌转移复发或预后预测的可行性，及其介导炎症因子TNFα诱导上皮间变而调控胆管癌侵袭转移的信号机制。

炎性微环境诱导并促进肿瘤侵袭转移是近年来关注热点。前期研究我们发现四跨膜蛋白CO-029表达可促进肝内胆管细胞癌侵袭转移，并首次发现炎性因子TNFα之受体--TNFαR1可与CO-029形成复合物，且干扰CO-029后TNFα诱导的胆管癌上皮间质转化（EMT）被阻断。本研究中我们首先在胆管癌细胞系（不同转移潜能的）和374例肝胆管细胞癌根治切除术后临床大样本（Cohort 1=120, Cohort 2=254）中发现CO-029过表达与胆管癌侵袭转移正相关；大样本（Cohort 2=254）COX模型预后分析我们发现CO-029高表达组肝胆管细胞癌术后5年生存率（Overall survival，OS）较CO-029低表达组胆管癌显著缩短（P=0.021），同时术后转移复发时间（Time to recurrence，TTR）相比CO-029高表达组胆管癌较低表达组胆管癌亦显著缩短（P=0.011）。进一步我们应用RNA干扰技术（CO-029 depletion）行体内外实验研究显示，CO-029高表达的胆管癌细胞HCCC-9810 和对照组HCCC-9810-Mock(LV-GFP)较CO-029-RNA干扰的HCCC-9810-shRNA-Co-029的细胞在体外增殖、侵袭、体内成瘤生长及肿瘤肺转移等方面明显增强（P<0.05, respectively）。深入机制研究发现：慢病毒干扰CO-029和（或）TNFαR1表达后发现CO-029与TNFαR1在功能上密切相关，TNFα的作用下CO-029高表达癌细胞增殖和侵袭能力显著增强，而CO-029干扰组未见改变；同时，CO-029高表达癌细胞HCCC-9810发生EMT，而在干扰组HCCC-9810-shRNA-Co-029细胞中未见发生，提示CO-029表达干扰后TNFα促癌细胞EMT的作用被抑制，CO-029结合TNFαR1后可诱导胆管癌EMT而促进侵袭转移。结论：四跨膜蛋白CO-029与肝胆管癌侵袭转移正相关，并可与TNFαR1结合而诱导癌细胞发生EMT改变而促进转移，是肝胆管癌转移复发及预后干预的潜在靶点和分子靶向治疗指标。