新抑癌基因SCNN1B在胃癌中的功能机制和临床应用价值研究

Employing genome-wide DNA methylation array analysis, we have identified SCNN1B as a novel gene methylated in gastric cancer cells compared to normal gastric tissues. Methylation-specific PCR (MSP) and bisulfite sequencing (BGS) validated SCNN1B promoter methylation in gastric cancer tissues and cell lines, which is associated the silencing of SCNN1B expression. Re-expression of SCNN1B in AGS cells inhibited cell proliferation, induced cell cycle arrest and apoptosis in vitro, suggesting that SCNN1B is a functional tumor suppressor in gastric cancer. Preliminary pathway analysis revealed that RAF-MEK-ERK pathway is a main target of SCNN1B. We therefore hypothesize that SCNN1B is a tumor suppressor that directly binds to RAF1 and regulates its activation, which in turn, inhibits the RAF-MEK-ERK signaling cascade and its downstream factors that mediate cell survival and metastasis. In this study, 1) we will assess the functional significance of SCNN1B in gastric cancer in vitro and in vivo. SCNN1B will be over-expressed or knockdown in gastric cancer cell lines and their effect on cell growth and metastasis will be examined in culture or in nude mice models. 2) We will determine the molecular mechanism of RAF-MEK-ERK inhibition by SCNN1B. The effect of SCNN1B on RAF1 dimerization, phosphorylation and kinase activity will be assessed by co-immunoprecipitation, western blot, and RAF1 kinase assay, respectively. Signaling through MEK-ERK will be examined by western blot analysis of its phosphorylation, and the activation of its downstream transcription factors will be determined by dual luciferase reporter assays. 3) Finally, we will investigate the prognostic significance of SCNN1B promoter methylation and protein expression in a cohort of gastric cancer patients (>200 samples). Patient samples have already been collected in our tissue bank, and we will use BGS and immunohistochemistry on tissue microarray to determine DNA methylation and protein expression, respectively and analyze their correlation with clinicopathological parameters. This study will provide important evidence for the functional significance, mechanism of action and clinical implication of SCNN1B in gastric cancer.

在我国，胃癌死亡率居癌症死亡前列。应用甲基化芯片进行全基因组扫描，我们找到新的潜在抑癌基因SCNN1B。该基因在胃癌细胞中沉默表达，而在正常胃组织中高表达。此外，在胃癌细胞株中过表达SCNN1B基因显著抑制细胞增殖，诱导细胞凋亡和周期阻滞。上述前期结果提示SCNN1B是胃癌新的肿瘤抑制基因。在此基础上，本课题主要研究：1）通过体内和体外实验并基于外源性过表达和shRNA靶向干扰技术探索SCNN1B对胃癌生长的作用；2）探讨SCNN1B对胃癌抑制的分子机制，重点阐明SCNN1B如何通过RAF-MEK-ERK 信号通路抑制胃癌；3）通过大样本胃癌独立队列研究，分析SCNN1B基因的表达异常或甲基化与胃癌临床预后的关系，探讨该基因在临床预后判断中的应用价值。本项目的开展有助于进一步阐明胃癌发生发展的分子机制，证实SCNN1B是新的胃癌抑癌基因，并有望成为预测胃癌预后的新型分子标志物。

胃癌是目前世界上最常见的恶性肿瘤之一。由于胃癌不易早期发现，就诊时往往已经是中晚期，加之缺乏有效的治疗手段，胃癌仍然是严重影响人民生命健康的重要疾病。癌基因的激活和抑癌基因的失活在肿瘤的发生发展过程中发挥重要作用。抑癌基因表观遗传学的变化是导致肿瘤发生的重要机制，而DNA甲基化修饰是抑癌基因失活的主要原因之一，在胃癌发展的过程中起很重要的作用。为了识别更多新的抑癌基因，我们使用Infinium Human Methylation 450K甲基化芯片进行全基因组扫描，筛选出一组包括SCNN1B在内显著受甲基化调控的新型基因。与正常胃上皮细胞和组织相比，SCNN1B基因启动子区CpG岛在人类胃癌中表现为高甲基化。因而推测其极有可能在胃癌的发生发展中发挥抑癌基因的功能。进一步研究发现，SCNN1B基因在81.3%的胃癌细胞株（13/16）和胃癌组织（P<0.001）中表达沉默，而在人类正常胃组织中高表达。DNA启动子区高甲基化水平与SCNN1B的转录沉默相关。胃癌组织芯片检测结果显示SCNN1B高表达是预测胃癌患者更长疾病特异生存时间的预后因子（P=0.004）。在胃癌细胞中过表达SCNN1B能抑制细胞生长，诱导凋亡和G0/G1期细胞周期阻滞，抑制细胞迁移与侵袭，体内实验抑制裸鼠皮下移植瘤生长。蛋白SCNN1B与蛋白GRP78直接相互作用，促进泛素化途径介导的蛋白GRP78的降解。随后GRP78蛋白水平的下降引发折叠蛋白应答（UPR），顺序激活PERK、ATF4、CHOP和XBP1s，导致caspase诱导的细胞凋亡以及对细胞迁移和侵袭的抑制。过表达SCNN1B使胃癌细胞对UPR诱导剂衣霉素细胞毒性的敏感性增强，说明SCNN1B能通过UPR介导肿瘤抑制。补救实验中过表达GRP78抵消SCNN1B对细胞生长和转移的抑制，而敲低GRP78则加强了SCNN1B对细胞生长的抑制。总之，在胃癌中SCNN1B通过靶向GRP78蛋白、激活UPR及其下游效应物，是一种新型肿瘤抑制剂。SCNN1B的表达状态可作为判断胃癌患者生存预后的独立生物学标志物。本课题的开展将不仅有助于进一步阐明胃癌发生发展的分子机制，还将有助于开发能预测胃癌患者预后的新型肿瘤标志物，是当前胃癌分子靶标研究领域创新性课题。