基于外显子组测序技术的胃癌转移相关的ACVR2基因突变检测与功能鉴定

There’s limited knowledge about driver genes of gastric cancer, so as to be difficult in new drug development and effects promotion. Recently, we used whole-exome sequencing of gastric adenocarcinoma tissues with high metastatic treand to identifymetastasis related mutations, and found ACVR2A mutation. In vitro experiments showed that ACVR2A down-regulation by siRNA increased the proliferation and migration of gastric cancer cells. There’s no report of the function of ACVR2A mutataion in gastric cancer. We conceive that wild type ACVR2A may function as a tumor suppressor, while mutant ACVR2A gain a function as an oncogene and can promote metastasis in gastric cancer. In the present study, we’ll detect the mutation of ACVR2A in more gastric adenocarcinoma samples and analyze it’s correlation with metastasis and patientsprognosis. Furtherly, we’ll study the functions of wild type and mutant ACVR2A in regulating transformation of gastric mucosa cells, and EMT, metastasis and stemness of gastric cancer cells, and explore the mechanisms from down-stream SMAD and protein kinases signaling pathway, by in vitro cell experiment and in vivo animal model. Completion of this study will clarify the mutation status of ACVR2A and it’s function in gastric cancer, and will help us deeply understanding the mechanisms of carcinogenesis and development of gastric cancer, and may help to identify new driver genes and provide targets for treatment and new drug development.

胃癌的驱动基因所知有限，致使新药研发困难、疗效难以提高。我们前期采用全外显子测序技术在高转移倾向胃癌组织中筛选转移相关突变，发现其中较高频度的ACVR2A基因突变，细胞学实验采用siRNA下调ACVR2A可促进胃癌细胞的增殖与迁移。ACVR2A突变在胃癌中的作用尚无报道。我们设想野生型ACVR2A可能在胃癌中发挥抑癌作用，而突变后获得了癌基因的作用并促进转移。本项目拟扩大样本量检测胃癌组织中ACVR2A基因突变并分析其与胃癌转移、预后等的相关性；通过细胞学与动物实验，进一步阐明野生型、突变型ACVR2A对胃粘膜细胞的恶性转化、胃癌细胞的EMT与转移、干细胞样特性的调控作用，并从下游转录因子SMAD及蛋白激酶途径分析其机制。本项目的完成，将阐明ACVR2A在胃癌中的突变与生物学功能，有助于胃癌发生发展机制的深入理解并可能发现新的驱动基因、为胃癌的治疗与新药研发提供新靶点。

胃癌是常见的恶性肿瘤之一，且大部分发生在中国，复发转移是胃癌患者死亡的主要原因，寻找驱动胃癌转移的基因并探索其作用机制可能为胃癌的治疗提供新的靶点。我们采用全外显子测序技术对胃癌组织进行测序，并结合TCGA等公共数据库数据分析，筛选出了胃癌转移相关基因ACVR2A。ACVR2A在胃癌中具有较高的突变频率，且突变位点相对集中，大多为截断突变。进一步分析发现ACVR2A的高频突变位点为微卫星位点，ACVR2A的突变与微卫星高度不稳定及高肿瘤负荷相关，ACVR2A突变组患者淋巴结转移倾向下降，而MSI-H患者较MSI-L/MSS患者具有更低的淋巴结转移倾向。对野生型组及突变组免疫细胞浸润分析发现，突变组较野生型组具有较多的活化NK细胞浸润。体外实验证实过表达野生型ACVR2A可促进胃癌细胞的迁移，而过表达ACVR2A突变体对胃癌细胞的迁移能力没有影响。过表达野生型及突变型ACVR2A对胃癌细胞的增殖、周期、克隆形成等均无明显影响。机制研究发现过表达野生型ACVR2A能激活下游SMAD信号通路，促进上皮间质转化，而突变型ACVR2A失去了这一功能。因此我们认为MSI-H组胃癌具有更低的淋巴结转移倾向可能与ACVR2A突变有关。鉴于ACVR2A突变与MSI-H及高TMB相关，使得ACVR2A突变可能成为识别MSI-H患者及选择免疫治疗患者的潜在生物标志物。