新基因psiTPTE22-HERV在胃癌中甲基化的临床应用价值及其功能研究

psiTPTE22-HERV is a genuine novel gene identified by us and possesses potential tumor suppressive activity. Its role and clinical application value in gastric cancer remains to be elucidated. Further study on gastric cancer revealed that psiTPTE22-HERV was silenced in ten gastric cancer cell lines by promoter methylation. psiTPTE22-HERV was specifically down-regulated in gastric tumor tissues as compared with corresponding adjacent normal tissues. In concordance with transcriptional down-regulation, DNA methylation was detected in gastric tumor samples. Meanwhile, psiTPTE22-HERV was expressed at a high level in normal gastric mucosa without promoter DNA methylation. Importantly, ectopic expression of psiTPTE22-HERV in silenced cancer cells was found to suppress cell growth and modulate the expression of many genes involved in important biological processes, such as cell proliferation, cell cycle regulation, etc. Based on these findings we aim: 1) to understand the silencing process of psiTPTE22-HERV during gastric carcinogenesis by examining its transcription and methylation levels in samples of intestinal metaplasia, dysplasia and tumor; 2) to explore the clinical application values of psiTPTE22-HERV promoter methylation on a large-scale cohort of gastric tumor and plasma samples; 3) to investigate the functions of psiTPTE22-HERV on cell proliferation, apoptosis, cell cycle regulation, migration/invasion and tumorigenecity by both in vitro and in vivo studies after ectopic expression in silenced gastric cancer cells; 4) to elucidate the molecular mechanism by which psiTPTE22-HERV functions by comparing the genome-wide mRNA expression profiles of psiTPTE22-HERV-overexpressed and empty vector-transfected gastric cancer cells. The resulting knowledge of this study will provide a comprehensive understanding of the role of psiTPTE22-HERV in gastric carcinogenesis and the potential clinical application of its promoter methylation in non-invasive diagnosis and prognosis of gastric cancer.

psiTPTE22-HERV是申请者发现并命名的新的潜在抑癌基因, 研究显示，psiTPTE22-HERV在10个胃癌细胞系和胃癌组织中由于启动子DNA甲基化导致其表达沉默，但正常胃粘膜无甲基化并呈高表达。转染该基因显著抑制癌细胞生长，基因表达芯片揭示该基因在肿瘤中调控多个重要下游基因。根据此前期研究结果，本课题旨在进一步研究：1)肠化生、异型增生及胃癌等不同病变阶段psiTPTE22-HERV的表达及甲基化水平，揭示其甲基化诱导的沉默趋势；2)在大规模胃癌组织及血浆标本中检测psiTPTE22-HERV的甲基化水平，发现其临床价值；3)观察过表达/敲低该基因对胃癌细胞增殖、凋亡、周期、侵袭能力和裸鼠致瘤性等的影响，揭示其功能；4)分析并验证psiTPTE22-HERV的分子调控信号通路，阐明其作用的分子机制。该研究将全面揭示新基因psiTPTE22-HERV在胃癌中的临床意义和功能机制。

背景：psiTPTE22-HERV是申请人近年自主发现的定位于psiTPTE22基因附近并包含一段HERV序列的新基因，其启动子区GC含量高，其表达受DNA甲基化调控。本项目旨在揭示该基因在胃癌中甲基化异常的临床意义及其生物学功能。.结果：psiTPTE22-HERV在多种正常组织（包括胃粘膜）中均表达较高，而在胃癌标本及多种胃癌细胞中由于DNA甲基化而表达下调或沉默，去甲基化药物处理能重新诱导该基因表达。psiTPTE22-HERV的甲基化在正常到胃癌进展过程中逐渐升高，而mRNA水平随胃癌进展逐渐下降。胃癌细胞SGC7901及MKN45中过表达该基因显著抑制的细胞生长、集落形成、周期进展、迁移和侵袭能力，促进凋亡；MKN1细胞中敲低该基因表达起相反效果。过表达该基因显著抑制SGC7901细胞在裸鼠皮下的成瘤能力，在尾部静脉注射转移模型中也显著降低肝转移率。表达谱分析发现psiTPTE22-HERV通过下调胃癌细胞代谢而起抑癌作用。定量检测的结果显示psiTPTE22-HERV启动子区甲基化水平在胃癌中比癌旁标本显著增高，并在两组中均比正常胃粘膜显著高。多因素Cox模型分析结果显示胃癌标本中psiTPTE22-HERV启动子区甲基化是胃癌病人预后的一个独立危险因素；Kaplan-Meier生存曲线显示高甲基化水平与预后差显著相关。在66.0%胃癌病人血浆中检测到psiTPTE22-HERV启动子区甲基化，而只在3.3%正常人血浆中检测到（P<0.001），提示血浆检测该基因甲基化也能作为胃癌无创诊断新方法。.结论：psiTPTE22-HERV在胃癌中因启动子区甲基化而表达下调，在胃癌中起抑癌作用，其甲基化可作为新的胃癌预后及无创诊断标志物。