STIP1蛋白介导射频消融后肝癌复发转移的机制研究

The Radiofrequency ablation (RFA) is one of the most important therapies to treat the primary hepatocarcinoma. However, it has higher recurrence rate compared to surgery. Studies have shown that ablation can not eliminate tumor cells completely，and the residual tumor cells have greater ability to invade and metastasize. The key factor still remains unexplored. In our previous study, we found that incomplete ablation can enhance the expression of stress inducible protein 1, and promote the EMT and metastasis. Besides, we found that Stip1 can interact with HSP90 and Snail to form protein-complex, and transfer into nuclear to modulate the transcription of Metal Matrix proteinase. Through the experiments such as ChIP, SiRNA, and animal model of in situ incomplete ablation , this project aims to demonstrate that the incomplete ablation can increase the expression of Stip1 in the residual HCC cells to promote the tumor metastasis in vivo and in vitro, and to further study the mechanism of action among Stip1, HSP90 and Snail as well as the interaction domain of gene regulation. And finally it can indicate the molecular mechanism of high recurrence and metastasis after RFA and the potential therapeutic target, to lay the theoreticalfoundation to prevent the recurrence after ablation.

射频消融是治疗原发性肝癌最重要的手段之一，然而与手术切除相比复发率更高。研究证实消融难以保证完全消灭肿瘤细胞，而残留肝癌细胞具有更强侵袭转移能力,其中的关键分子事件仍不明确。我们前期研究发现肝癌细胞中不全消融可以诱导HSP90的辅助伴侣分子STIP1表达上调并促进肝癌细胞上皮间质转化和侵袭转移,且发现STIP1可以与HSP90以及SNAIL组成蛋白复合体进入核内调节金属基质酶的转录活性。本项目拟通过染色质免疫沉淀,siRNA干扰、原位肝癌不全消融动物模型等实验，在体内和体外试验中证实不全消融通过上调残余肝癌细胞STIP1表达促进肿瘤侵袭转移，进而深入探讨STIP1与HSP90及SNAIL的作用机制及调节基因的互作序列，揭示射频消融后肿瘤高复发转移的分子机制并寻找潜在的治疗靶点，为防治消融术后复发提供理论基础。

近年来的研究表明，不全射频消融（RFA）可使肝癌（HCC）具有更高的侵袭性。磷酸化应激诱导蛋白1（STIP1）在肝癌中高表达，是介导热应激下细胞内稳态的分子伴侣。我们旨在探讨STIP1在RFA后残留肝癌中的促转移作用。我们采用原位肝癌移植或尾静脉注射构建的肝癌小鼠模型，评价肝癌肺转移或肝内转移的可能性。细胞培养模型用于细胞侵袭实验、明确间充质特异基因的表达情况及其潜在的分子机制，同时，我们收集临床标本分析STIP1与临床预后的关系。我们发现不全RFA会导致HCCLM3肿瘤的肝内转移（IHM）增多，而通过沉默STIP1可以减少IHM。STIP1基因敲除也显著降低HCCLM3细胞的肺转移潜能。在体外，HCCLM3和HepG2在亚致死热暴露后呈现梭形形态，STIP1和间充质特异基因表达上调。热暴露诱导了STIP1热休克蛋白90（HSP90）复合物的形成，该复合物能促进上皮转录抑制因子Snail1入核并调节间充质基因转录。阻断HSP90-STIP1复合物可降低热暴露后肝癌细胞的侵袭潜能。通过临床标本分析，发现转移性肝癌组织和转移性肝癌患者血清中STIP1的表达明显增高（p<0.05）。STIP1的高表达与无复发生存期短有显著相关性（p<0.05）。综上所述，我们的研究发现，STIP1通过介导间充质基因转录并与亚致死热诱导的癌细胞转移呈正相关。阻断STIP1活性可能抑制RFA后肝癌细胞的转移潜能。