DNMT3A2通过lncRNA-TRERNA1-E-cadherin轴在胃癌侵袭转移中作用机制研究

Gastric cancer is a kind of malignant tumor which is highly invasive and metastatic and it is severely harmful to life. Epithelial-mesenchymal transition (EMT) is an important event in the progress of the metastasis of gastric cancer. Our research recently found that DNMT3A2 not only silenced E-cadherin via DNA methylation together with histone methylation, but also combined with lncRNA-TRERNA1 to regulate the expression of E-cadherin in the transcriptional and translational level, inducing gastric cancer EMT and promoting the invasion and metastasis of gastric cancer. REMSA and GST pull-down were applied to analyze the binding region of DNMT3A2 and lncRNA-TRERNA1 and its regulation of the expression of EMT related molecules, besides, the regulation of EMT-TFs by TRERNA1 was also detected. RNA-seq of DNMT3A2-RIP product was employed to explore other targeted lncRNAs of DNMT3A2 and its regulation of EMT-TFs in the level of cell model -animal model -clinical samples. Deep analysis of mechanism of DNMT3A2 in the invasion and metastasis of gastric cancer via lncRNA-TRERNA1-E-cadherin axis, and provided a theoretical basis and an experimental basis for the epigenetic intervention for the invasion and metastasis of gastric cancer.

胃癌是具有高度转移性并对生命造成严重危害的恶性肿瘤。上皮细胞间质转化（EMT）是胃癌发生转移过程中重要事件。本课题组近年研究发现DNMT3A2不仅以DNA甲基化及协同组蛋白甲基化作用沉默E-cadherin而诱导胃癌细胞EMT，且结合了lncRNA-TRERNA1在转录、翻译水平调控E-cadherin表达而促进胃癌细胞转移。本项目拟采用RIP,REMSA结合GST pull-down分析DNMT3A2与TRERNA1结合、结合区段及对E-cadherin表达调控, TRERNA1对EMT-TFs的调控及意义；RIP-RNA-seq分析DNMT3A2结合其他lncRNA 及对EMT-TFs的调控；于细胞模型-动物模型-临床样本层面，深入分析DNMT3A2通过TRERNA1-E-cadherin轴及lncRNA在胃癌侵袭转移中作用机制，为胃癌发生转移过程的表观遗传干预提供理论依据和实验基础。

DNA甲基转移酶3A (DNA methyltransferase 3A, DNMT3A)作为从头甲基转移酶在个体发育过程中对DNA甲基化模式的建立起到重要作用，其在胃癌中高表达与胃癌患者的预后低生存率具有显著相关性，然而DNMT3A介导胃癌发生发展的调控机制尚不清楚。DNMT3A基因能够编码产生两种具有甲基转移酶活性的isoform, 分别是全长形式的DNMT3A1和截短形式的DNMT3A2，它们在机体细胞中具有不同的表达谱和亚细胞定位。本项目前期研究发现，通过研究发现DNMT3A2不仅以DNA甲基化及协同组蛋白甲基化作用沉默E-cadherin而诱导胃癌细胞Epithelial Mesenchymal Transition (EMT)，且结合了lncRNA-TRERNA1调控E-cadherin表达而促进胃癌细胞转移。本项目通过研究进一步揭示DNMT3A1和DNMT3A2在胃癌组织中呈现高表达模式，但在胃癌发生发展中参与的生物学功能和调控机制有所不同。DNMT3A1调控细胞周期相关分子CDK4和CDK6的蛋白表达促进胃癌细胞恶性增殖；而DNMT3A2通过作用EMT相关标志分子影响胃癌细胞恶性转移特征。DNMT3A2在胃癌组织中呈现高表达率为65% ，且DNMT3A2的高表达与胃癌的TNM分期和淋巴结转移存在显著性相关； DNMT3A2不仅通过DNA甲基化水平作用抑制上皮样细胞标志分子E-cadherin和β-cantenin的表达， 同时调控EMT标志物分子EpCAM、Claudin7、FN1和MMP7的表达。本研究进一步证明DNMT3A2与lncRNA TRERNA1结合，通过TRERNA1增强子样作用激活了SNAI1转录，进而抑制CDH1的表达，促进胃癌细胞侵袭和转移；我们研究发现，DNMT3A能够与TRERNA1结合，并结合EZH2，TRERNA1绑定EZH2使CDH1基因启动子区H3K27me3增加从而抑制CDH1的转录；TRERNA1的表达受转录因子TFAP4的调控，TFAP4通过直接结合于TRERNA1启动子区的E-box调控TRERNA1的表达。本研究结果不仅证明DNMT3A2通过作用EMT相关标志分子影响胃癌细胞恶性转移特征，同时揭示了TRERNA1可以绑定DNMT3A2且受TFAP4调控，为胃癌临床的早期诊断和辅助治疗提供了新的思路。