基于外显子测序技术筛选胃癌复发转移新基因CDH4突变及其功能研究

In addition to the poor effect of chemotherapy, little understanding of the driver genes in gastric cancer is the main reason for the short survival time of recurrent and metastatic gastric cancer patients. To find the key driven-genes is essential for making a significant breakthrough on targeted therapy. Our previous study showed that CDH4 gene mutation was up to 10.4% in recurrence group, but only 2.6% in relapse-free group. We further found that CDH4 knock-down promoted metastasis and chemotherapy resistance by undergoing epithelial-mesenchymal transition (EMT). Therefore, we speculated that CDH4 mutation might participate in the metastasis and chemotherapy resistance of gastric cancer through EMT process. In this study, we planned to detect the frequency and sites of CDH4 gene mutations by enlarging the sample size of gastric cancer tissues and analyze the correlation between the mutation sites and protein expression, metastasis and prognosis, respectively, and to further analyze the variant protein and cell function alteration after CDH4 mutation, and identify the functional changes (loss of function or gain of function) with the use of clinical data. The activation or silencing of CDH4 downstream signaling pathway after mutation is also an important research task. This project aims to further illustrate the CDH4 mutation rate and variant protein expression, biological function and regulatory mechanisms, providing a new target for the development of new drugs, which has potential application values.

复发转移胃癌的生存期很短，除了化疗效果差外，主要原因是对胃癌驱动基因了解甚少，导致多数靶向药物无用武之地。寻找关键驱动基因是突破靶向治疗瓶颈的重要举措。前期我们通过全外显子测序发现CDH4基因突变率在复发组高达10.4%，而非复发组仅2.6%；野生型CDH4表达下调可能通过上皮间质转化（EMT）促进转移和化疗耐药。因此，我们推测CDH4突变可能通过EMT作用介导胃癌复发转移及化疗耐药。本研究拟通过扩大样本量明确胃癌中CDH4突变频率与位点，并分析其与蛋白表达、转移与预后的相关性；进一步分析突变后的变异蛋白与细胞功能的改变，结合临床数据，明确这些基因位点突变所带来的功能改变（丢失或获得功能）。CDH4下游的信号通路在突变后的激活或沉默状态也是本课题的重要研究内容。本项目旨在深入阐明CDH4在胃癌组织中的突变与变异蛋白表达情况、生物学功能以及机制，为新药研发提供新的靶点，具有潜在的应用价值。

胃癌是我国最常见的恶性肿瘤之一，胃癌根治术后发生复发转移是胃癌患者死亡的主要原因。基因突变在胃癌复发转移中扮演重要角色。因此，寻找胃癌复发转移的驱动基因显得尤为重要。我们采用全外显子测序技术对胃癌组织进行测序，结合TCGA、PolyPhen-2在线数据库筛选出胃癌复发转移相关的功能基因CDH4。我们发现CDH4在胃癌复发组中的突变率较未复发组升高。进一步收集临床资料，生存分析证实CDH4在胃癌突变患者中的总生存时间（OS）和无疾病生存时间（DFS）较野生型有缩短趋势，提示CDH4突变与胃癌患者的不良预后相关。TCGA数据库分析提示CDH4突变与TMB、MSI score、临床分期相关，同时TIMER数据库分析CDH4表达与免疫细胞浸润显著相关。CCK-8和Tranwell实验提示野生型CDH4抑制胃癌细胞增殖、迁移；IC50实验提示野生型CDH4增加胃癌细胞对5-氟尿嘧啶化疗敏感性。突变型CDH4可逆转上述功能。通过RNA-Seq进行机制研究筛选出与CDH4发挥生物学功能有关的下游基因，并通过Real-time PCR进行验证。综上所述，我们认为CDH4突变可能作为新的化疗及免疫治疗的疗效预测指标，并与胃癌患者的不良预后密切相关。