PADI4协同IL-8/CXCR2通路促进胃癌进展的分子机制研究

Inflammatory factors could promote gastric cancer cell proliferation, migration and angiogenesis, and the disregulation of IL-8/CXCR2 pathway is closely related with the occurrence and development of gastric cancer. Peptidylarginine deiminase 4 (PADI4) could promote tumor progression through post-translationally modifying arginine into citrulline. Our previous study found that PADI4 was highly expressed in the tissue and serum of gastric cancer and could promote the progression of gastric cancer. However, the underlying mechanism is still obscure. Recently, we found that 1) PADI4 could regulate the expression of CXCR2 and IL-8 protein; 2) PADI4 could be induced in the presence of inflammation factor IL-8, indicating that PADI4 may promote the progression of gastric cancer through regulating the IL-8/CXCR2 pathway. In this study, we applied of various techniques, such as gene transfection and inhibitor blocking in order to: (1) elucidate the regulation of PADI4 on the expression and function of IL-8 and CXCR2; (2) study the effect of PADI4 on the biology activity and affinity of IL-8/CXCR2; (3) characterize the expression PADI4 under inflammatory condition, such as IL-8;(4) characterize the clinico-pathological features of PADI4, IL-8 and CXCR2.The purpose of this study is to study the coordination between PADI4 and IL-8/CXCR2 pathway during tumor progression, and analyze whether PADI4 could be treated as a "inflammatory regulator factor" promote the progress of gastric cancer. This study may reveal a novel pathological mechanism regarding gastric cancer and provide credible evidence to treat gastric cancer with the inhibitor of PADI4.

炎症相关因子可促进胃癌细胞的增殖，迁移和血管生成等，其中IL-8/CXCR2通路异常与胃癌相关。PADI4通过将蛋白的精氨酸催化为瓜氨酸，参与肿瘤病变，我们首次发现PADI4在胃癌组织和血清中高表达，可促进胃癌的发生，但致病机制不清。最近我们研究发现PADI4与IL-8/CXCR2之间存在相互调控作用，提示PADI4可能通过调控IL-8/CXCR2通路促进胃癌进展。本课题拟利用基因转染和抑制剂阻断等方法，明确1)PADI4对IL-8/CXCR2表达和功能的调控；2)PADI4对IL-8/CXCR2活性和亲和力的影响；3)炎性因子IL-8对PADI4的表达调控；4)PADI4、IL-8和CXCR2在胃癌组织中的表达相关性，探讨PADI4协同IL-8/CXCR2通路促进胃癌进展的分子机制，分析PADI4是否可作为“炎性调控因子”促进胃癌进展，为靶向PADI4抑制剂的临床治疗提供新的依据。

胃癌由于其较高的转移性，是目前世界上致死率最高的癌症之一，我们前期发现肽基精氨酸脱亚胺酶4（PADI4）是胃癌的易感基因，本课题通过在胃癌细胞中转染过表达PADI4质粒的方法，发现过表达PADI4后能显著增加胃癌细胞的增殖，迁移，克隆形成能力，裸鼠成瘤能力，并降低细胞的凋亡能力。炎性因子在胃癌的发生和发展过程中发挥重要作用，因此，我们采用多组分炎性ELISA试剂盒的方法，发现过表达PADI4后炎性因子IL-8的表达量明显增高，我们通过小分子RNA干扰IL-8的表达，发现抑制IL-8的表达后能明显的抑制过表达PADI4后产生的迁移能力。说明PADI4在促进胃癌细胞的迁移能力可能是通过调控IL-8的表达产生的。我们又采用IL-8蛋白刺激胃癌细胞，发现IL-8可以通过MAPK通路调控PADI4的表达。以上结果说明PADI4协同IL8参与胃癌的进展。本课题组同时研究了PADI4对食管癌发病机制的研究，发现转染过表达PADI4质粒后，食管癌细胞的增殖和肿瘤形成能力明显的增高，并调控CA9的表达，进而促进肿瘤的生长和转移。说明PADI4可以作为胃癌，食管癌的治疗靶点。我们采用同样的方法检测了PADI4的同家族蛋白PADI3在结肠癌中的功能，结果显示，PADI3在结肠癌细胞中与PADI4发挥相反的作用，在结肠癌细胞中过表达PADI3后，结肠癌细胞出现增殖和克隆形成能力下降，G1期阻滞现象等。我们又采用转录组测序分析PADI3在结肠癌肿瘤发生中的分子机制，结果显示PADI3能显著降低Sirt2，Snail的表达水平以及AKT的磷酸化水平，并能显提升p21的表达水平，并且过表达Sirt2能部分逆转PADI3过表达对细胞造成的影响。免疫细胞化学实验显示，PADI3主要定位于细胞质中。最后我们用缺失突变实验用于确定PADI3发挥功能的关键结构域，结果发现C-结构域是PADI3发挥抗癌功能的关键结构域。本研究组最新研究发现PADI3与CKS1(细胞周期依赖的蛋白激酶调节亚基1)呈负相关，过表达PADI3能在一定程度上抑制CKS1诱导的癌细胞增殖。进一步研究发现，PADI3能显著抑制Hsp90和CKS1表达，并且Hsp90对于PADI3在结肠癌细胞中抑制CKS1的表达是必须的，说明PADI3蛋白下一步可开发为治疗结肠癌的药物。