WWOX基因遗传变异影响胃癌患者预后的分子机制研究

Human WWOX gene is located on chromosome 16, and its coding protein can inhibit autophagy and maintain genomic stability by activating mTOR and DNA damage repair protein, thus affecting the development of tumors. Previous studies have shown that genetic variation is one of the important factors leading to the difference in prognosis of gastric cancer patients, which can be used as a tool for early prognosis prediction. Based on the population including 1002 gastric cancer patients in China, we found that WWOX rs74684850A>G significantly associated with prolonged overall survival of patients and WWOX mRNA expression, but the molecular mechanism underlying this association was unclear. Bioinformatics analysis suggested that this SNP was involved in the formation of the WWOX gene enhancer, and that A>G might enhance the binding of DNA fragments to the transcription factor ZIC1. Cell experiments revealed that WWOX gene acted as a tumor suppressor in gastric cancer and inhibited the expression of GCNT3, one of the key proteins in the mucin type O-glycan biosynthesis pathway. Based on these initial results, we hypothesize that this variant may regulate the expression of WWOX gene and its downstream mucin type O-glycan biosynthesis pathway via interaction with ZIC1, thereby affecting the prognosis of patients with gastric cancer. We next will systematically elucidate the detailed mechanism through the experiments with gastric cancer cells, animal models and clinical samples. The successful implementation of this project can provide new molecular marker for early survival supervision and assist the development of individualized treatment strategies for patients with gastric cancer.

人类WWOX基因位于16号染色体，其编码蛋白可通过激活mTOR及DNA损伤修复蛋白，抑制细胞自噬及维持基因组稳定性，进而影响肿瘤的发展。遗传变异是导致胃癌患者预后差异的重要因素之一，可辅助预后的早期预测。本项目基于中国胃癌患者人群1002例，前期发现WWOX rs74684850A>G遗传变异显著延长患者总生存期，且与WWOX mRNA表达量相关，但其中的具体机制尚不明确。生物信息分析提示该遗传变异可能参与构成WWOX基因增强子，A>G可能增强DNA片段与转录因子ZIC1结合。细胞实验揭示WWOX基因在胃癌中起抑癌作用，且可抑制黏蛋白O-型糖链合成通路关键蛋白之一GCNT3的表达。后续拟通过细胞、动物实验及临床标本检测，系统阐明该遗传变异通过ZIC1调控WWOX基因表达及其下游黏蛋白O-型糖链合成通路，影响胃癌患者预后的具体机制，为患者早期生存监测提供新的分子标记物，协助个体化治疗。

既往研究报道WWOX基因可能通过mTOR和DNA损伤修复通路来影响肿瘤的发展。我们既往报道了mTOR和DNA损伤修复通路相关单核苷酸多态性（Single nucleotide polymorphism, SNPs) 和胃癌患者的预后相关，因此我们假设WWOX基因遗传变异可能预测胃癌患者的预后。通过WWOX相关遗传变异位点进行候选策略的检测，我们发现WWOX基因相关遗传变异和胃癌患者的预后密切相关，且发现一内含子位点位于潜在增强子区域。基因型-表型关联分析发现该变异位点可影响WWOX基因的表达。进一步分析发现该位点在胃癌细胞中和WWOX基因相关启动子距离远，因此进一步探索相关分子机制。进一步细胞实验发现，发生该位点变异后可在荧光素酶报告质粒中抑制Luciferase基因的转录活性，且和位点的DNA方向无关，该Luciferase基因是被WWOX启动子所驱动。由于该位点位于潜在增强子区域，因此该位点可能通过顺势作用调控机制和WWOX启动子互作从而影响WWOX基因的表达。EMSA实验发现该变异可降低NR3C1转录因子的结合能力，且Western Blot实验发现NR3C1可促进胃癌细胞中WWOX蛋白的表达。最后通过细胞功能实验，发现敲减WWOX基因可以促进胃癌细胞的侵袭、迁移和增殖。本研究奠定了基因遗传变异顺式调控WWOX基因表达进而影响胃癌患者预后的分子机制，有助于胃癌患者的个体化治疗。