LOXL2调控胆管癌细胞Snail的过程及机制研究

Zinc finger transcription factor Snail is key gene to modulate the procession of EMTthrough repression of E-cadherin either by direct or indirect mechanisms.Recent findings indicating that Lysyl-oxidase like 2 enzyme (LOXL2) is a new player in the regulation of EMT. LOXL2 promotes EMT through enhance stability of Snail.However,the mechanism of LOXL2 regulate Snail is still unclear.In our past experiments,we found the expression of Snail, SUMO and Ubc9 increased in cholangiocarcinoma cell lines after upregualtion of LXOL2; At same time, Snail was found to be contact with SUMO in CCA cells by co-immunoprecipitation experiment. Based on the above data,we hypothesis that "LOXL2 regulate Snail by SUMOylation pathway in CCA cells". We plan to detect the exprseeion of Snail,SUMO and Ubc9 after regulation of LOXL2, SUMO in CCA cell lines by Co-immunoprecipitation,Western blot and Cycloheximide chase. The aims of the project is to study deeply the mechanism of LOXL2 regulation to Snail and provide the potential target for prevention and treatment of invasion and metastasis of tumor.

Snail是介导EMT发生的关键因子，其通过沉默E-cadherin的表达进而调控EMT的发生发展。赖氨酰氧化酶样2基因（LOXL2）是新发现的EMT关键调控因子。LOXL2通过提高Snail的稳定性而抑制E-cadherin的表达。但是有关LOXL2调控Snail的机制尚缺乏深入研究。我们前期实验发现上调胆管癌细胞LOXL2的表达伴随Snail、SUMOylation蛋白SUMO及Ubc9的表达上调；免疫共沉淀发现SUMO与Snail结合表达。根据前期工作基础，我们提出"LOXL2通过促进苏素化水平，抑制Snail的降解"的假说。本课题拟通过调控胆管癌细胞LOXL2、SUMO的表达及诱导Snail K98和K137点突变，采用免疫共沉淀及放线菌酮追逐实验等技术，检测Snail的稳定性、SUMO及Ubc9的表达，深入研究LOXL2调控Snail的机制，为预防及治疗肿瘤转移提供新的靶点。

已经报道了LOXL2与Snail的直接核相互作用和E-钙粘蛋白的下调以刺激上皮至间质转化（EMT），其促进癌症转移，但迄今尚未报道LOXL2调节Snail的证据。在这项研究中，我们证明LOXL2过表达调节胆管癌细胞中EMT相关分子的表达。此外，作为EMT关键调节因子的Snail1在蛋白质水平而不是mRNA水平受LOXL2调节。使用Co-IP实验，我们发现Snail1可以通过LOXL2过表达的小泛素样修饰物（SUMO2 / 3）外观进行修饰，而SENP的上调或SUMO的敲低消除了Snail的表达。将Snail1赖氨酸残基98突变为精氨酸（LOXL2 / Snail合作的位点）并未改变Snail的SUMO化。在侵袭性胆管癌组织中观察到LOXL2，Snail和Sumo2 / 3的免疫组织化学表达增加。这些研究结果表明，LOXL2以SUMO化的形式参与了胆管癌进展中Snail1的翻译后修饰。