DNMT3A表观修饰miR-338-3p通过HHAT和Rab14抑制胃癌侵袭转移的作用机制

The epigenetic changes are involved in the invasion and metastasis which are significant features of gastric cancer. Our previous study found that miR-338-3p is decreased and DNMT3A is up regulated together with TCGA database analysis, the negtive relationship was observed between miR-338-3p and DNMT3A in gastric cancers, and the expression of miR-338-3p was increased in gastric cancer cells treaed with 5'-Aza-dC.Overexpression of miR-338-3p can markedly inhibit the migration of gastric cancer cells. The target relationship between miR-338-3p and metastasis-associated HHAT/Rab14 is predicted using bioinformatics. Taken together, we propose that DNMT3A epigeneticly regulates the expression of miR-338-3p to inhibit invasion and migration through co-targeting HHAT and Rab14 in gastric cancer cells. In this study, we will apply immunohistochemistry and western blot to evaluate the clinical significance between gastric cancer patients and DNMT3A - miR-338-3p - HHAT/Rab14 axis. Futhermore, CHIP, qRT-PCR and reporter gene analysis are applied to analyze the effects of DNMT3A - miR-338-3p - HHAT/Rab14 axis on mechanism of invasion and metastasis of gastric cancer. Finally, tumor-bearing mouse model will be performed. Therefore, our research will provide new understanding to elucidate the mechanism of dysregulation of miR-338-3p with the invasion and metastasis of gastric cancer and potential clinical targeted therapy.

侵袭和转移是胃癌的重要特征，涉及表观遗传学改变。我们前期工作结合TCGA数据库分析发现在胃癌中miR-338-3p低表达，DNMT3A高表达，两者呈显著负相关，且DNA甲基化抑制剂5’-Aza-dC处理胃癌细胞引起miR-338-3p表达上调；过表达miR-338-3p可显著抑制胃癌细胞的迁移，生物信息学预测其靶向HHAT和Rab14，后二者与肿瘤侵袭转移密切相关，提示DNMT3A表观调控miR-338-3p的表达并通过HHAT和Rab14双靶点影响胃癌侵袭转移。本研究拟应用免疫组化、Western Blot等方法研究DNMT3A─miR-338-3p─HHAT/Rab14轴与胃癌患者的临床相关性；CHIP、qRT-PCR、报告基因、荷瘤小鼠模型等方法在细胞和动物水平系统分析该轴在胃癌侵袭转移中的作用机制。为阐明miR-338-3p在胃癌侵袭转移中的失调机制及寻找潜在治疗靶点提供新的认识。

胃癌是世界范围内常见的恶性肿瘤之一，位居我国恶性肿瘤发病率第2位，死亡率位居第3位。陕西省恶性肿瘤呈现地域化特点，胃癌在陕北地区高发。多数研究认为致癌基因或者抑癌基因的异常失调可能受到甲基化或miRNAs的调控所致。申请者前期工作中发现miR-338-3p在胃癌组织和细胞中表达下调，过表达miR-338-3p可显著抑制胃癌细胞的迁移侵袭能力，DNA甲基化抑制剂5’-Aza-dC处理胃癌细胞引起miR-338-3p表达上调，生物信息学预测其靶向HHAT和Rab14，后二者与肿瘤侵袭转移密切相关。课题基本按照原有计划进行，主要研究目标是阐明DNMT3A表观调控miR-338-3p的表达并通过HHAT和Rab14双靶点影响胃癌侵袭转移的分子机制，并分析其与胃癌患者的临床相关性。课题在细胞水平、动物水平和临床标本等不同层次解析了DNMT3A─miR-338-3p─HHAT/Rab14轴在胃癌侵袭转移中的作用机制。miR-338-3p在胃癌组织和细胞系中表达下调，其表达与胃癌的G分期密切相关，与生存率负相关；miR-338-3p与Rab14和HHAT有良好的靶向关系；体内体外实验均证实了miR-338-3p可通过抑制E-Cadherin蛋白的内吞作用和HHAT-SHH-AKT-MMP信号通路发挥其对胃癌细胞侵袭迁移的抑制；利用BSP-PCR对miR-338-3p启动子区CpG岛甲基化水平分析，5’-Aza-dC处理后，miR-338-3p第二个CpG岛启动子区甲基化水平由97.4%下降到71.5%；通过CHIP等研究方法分析DNMT3A对miR-338-3p的宿主基因AATK的调控机制及与胃癌临床指标的相关性，DNMT3A在胃癌组织和细胞中高表达，且与AATK表达显著负相关，AATK启动子区3个CpG位点（cg03809847、cg20541344、cg05292376）的甲基化水平与DNMT3A正相关，AATK表达下调与其启动子区CpG岛超甲基化水平有关，DNMT3A表观遗传修饰引起AATK的低表达。项目研究成果将为胃癌的转移提供新的认识，为胃癌靶向药物开发提供新的思路和策略。课题在执行期间共发表论文12篇，其中SCI11篇；参加国内学会会议5人次、省内学术会议10人次；培养硕士研究生5人。