新融合基因DDX5-POLG2在胃癌中的功能机制及临床价值研究

Transcription-induced fusion genes play important roles in cancers as shown by more and more studies. We have recently identified a new transcription-induced fusion, DDX5-POLG2, in gastric cancer cell lines by transcriptome sequencing. Further validation revealed that this fusion was expressed in 10 of the 13 tested gastric cancer cell lines and not detected in normal stomach mucosa. Fusion transcript of DDX5-POLG2 was detected in 47.8% of gastric tumor tissues (n=113) and 28.1% of adjacent normal tissues (n=96); the occurrence rate of DDX5-POLG2 expression was significantly higher in gastric tumor tissues than in adjacent normal tissues (p=0.04). Knockdown of DDX5-POLG2 significantly inhibited growth of gastric cancer AGS cells. Based on these findings we aim: 1) to understand the induction process of DDX5-POLG2 fusion during gastric carcinogenesis by examining its transcription and protein levels in samples of intestinal metaplasia, dysplasia and tumor, and explore the clinical application values of DDX5-POLG2 on a large-scale cohort of gastric tumor samples; 2) to investigate the functions of DDX5-POLG2 on cell proliferation, apoptosis, cell cycle regulation, migration/invasion and tumorigenecity by both in vitro and in vivo studies after ectopic expression or knockdown of DDX5-POLG2 in gastric cancer cell lines; 3) to determine the intracellular localization of the fusion protein, and elucidate the molecular mechanism of DDX5-POLG2 in regulating gene transcription or mitochondrial DNA replication, its direct interacting partners and its regulated downstream key cancer pathways. All investigation will be conducted in comparison with DDX5 and/or POLG2 to comprehensively understand the specific importance of the induction of DDX5-POLG2 fusion during gastric carcinogenesis. The resulting knowledge of this study will provide a comprehensive view of the clinical and functional significance of DDX5-POLG2 in gastric cancer.

转录诱导的融合基因在肿瘤中有重要作用。我们转录组测序发现胃癌中转录诱导的新融合基因DDX5-POLG2，验证发现其在10/13胃癌细胞系表达而正常胃粘膜不表达，胃癌组织中表达频率比癌旁显著高(47.8% vs 28.1%，p<0.01)；敲低该融合基因显著抑制胃癌细胞生长。根据此前期研究结果，本课题旨在：1）阐明在肠化生、异型增生及胃癌等不同病变阶段该融合基因的表达变化，并在大规模胃癌标本中研究其表达在诊断分期、预后判断等方面的临床意义；2）通过细胞增殖、凋亡、周期、侵袭能力和裸鼠致瘤性等实验揭示其功能;3）明确融合蛋白的细胞定位，在调控基因转录或线粒体DNA复制、相互作用蛋白及对重要信号通路调控等方面，深入阐明其作用机制。各项工作同时与DDX5及POLG2的表达及功能机制进行比较研究，以明了该融合基因表达的意义。该研究将全面揭示新融合基因 DDX5-POLG2在胃癌的临床意义和功能机制。

背景： DDX5-POLG2是申请人近年基于胃癌细胞转录组及全基因组测序自主发现的一个新融合基因，该融合基因的mRNA包含了除DDX5最后一个外显子与POLG2第一个外显子以外的所有外显子，编码相应的由DDX5和POLG2组成的融合蛋白并保留了最重要的功能保守域。本项目旨在深入阐明DDX5-POLG2在胃癌中的功能机制及临床意义。.结果：DDX5-POLG2在大部分正常组织样本(包括胃粘膜)中不表达，而在多个胃癌细胞系和在大规模胃癌标本中表达。DDX5-POLG2在胃癌中检出率(47.8%)比在癌旁正常标本(28.1%)中显著更高（P=0.004）。Kaplan-Meier生存曲线分析显示DDX5-POLG2融合基因的病人的生存期显著更短（P=0.015）。胃癌细胞AGS和MKN-28中敲低DDX5-POG2的表达显著抑制细胞生长与集落形成能力。胃癌细胞中过表达DDX5、POLG2以及DDX5-POLG2均能促进细胞生长与集落形成能力。GES-1和SNU-1中过表达DDX5-POLG2比DDX5及POLG2更为显著地提高了细胞活力、集落形成、迁移和侵袭能力。Western blot证实DDX5蛋白及POLG2蛋白定位于细胞核，而DDX5-POLG2新融合蛋白也定位于细胞核。RNA测序分析显示SNU-1细胞中DDX5-POLG2融合基因的表达相较于DDX5、POLG2表达或者空载体转染，均显著激活了MAPK、PI3K-Akt、TNF signaling及Complement and coagulation cascades通路。这些信号通路均对胃癌的进展及转移中起重要作用。我们发现了一个在DDX5-POLG2融合基因转染组特有的新融合基因LRRC42-HSPB11。.结论：DDX5-POLG2融合基因在胃癌细胞和胃癌样本中高表达，在胃癌发展中起促进作用，对胃癌的预后判断具有重要的临床应用价值。