新发现circRNA分子在幽门螺杆菌介导的胃黏膜细胞恶性转化中的作用及分子机制研究

Gastric cancer is one of the most common malignant tumors which affect the human beings' health seriously. Helicobacter pylori infection is an important initiating factor in the development of gastric cancer. However, the specific mechanism of the malignant transformation of gastric mucosal cells mediated by H. pylori infection has not yet been elucidated. The applicant for the first time through high-throughput RNA sequencing (RNA-Seq) technology and cellular and molecular biology experiments screen and validate a newly discovered circular RNA molecule circTVAS5 which is activated and plays important roles in the process of H. pylori related malignant transformation. Based on our previous work, this project will further investigate the following contents :(1)To elucidate the mechanism of circTVAS5 in post-transcriptional regulation of target genes as miRNA sponge in the cellular and molecular level; (2)To determine the association of circTVAS5 and gastric mucosa malignant transformation and the intervention effect of inhibiting circTVAS5 expression in gastric cancer development in vivo; (3)To detect the expression of circTVAS5 in the specimens of human gastric tissue and serum and determine the association of circTVAS5 and the malignant transformation of gastric mucosa mediated by H. pylori infection, and evaluate the potential value of circTVAS5 as a serum biomarker for early detection of gastric cancer. Through the above-mentioned research, we aim to explicate the role and molecular mechanism of circTVAS5 in the gastric mucosa malignant transformation induced by H. pylori infection, and to provide new ideas and novel strategies for the development of circTVAS5 as a molecular biomarker for early detection and the intervention target of gastric cancer.

胃癌是严重影响人类健康的恶性肿瘤之一，幽门螺杆菌感染是胃癌发生的重要始动因素，然而幽门螺杆菌感染介导胃黏膜细胞恶性转化的具体机制尚未阐明。申请者首次通过高通量RNA测序和细胞分子生物学实验筛选并验证出新发现的环状RNA分子circTVAS5在幽门螺杆菌感染向恶性转化过程中被激活并具有生物学功能。本项目将在前期工作基础上，进一步研究以下内容：（1）在细胞和分子水平解析circTVAS5作为miRNA海绵在转录后水平调控靶基因表达的分子机制；（2）在动物整体水平确定circTVAS5与胃黏膜恶性转化的关联及抑制circTVAS5表达对胃癌发生的干预作用；（3）在人体胃组织标本和血清中检测circTVAS5的表达，明确circTVAS5与幽门螺杆菌介导的胃黏膜细胞恶性演变的关联及其作为胃癌早期预警血清标志物的潜在价值。从而系统地阐述circTVAS5在幽门螺杆菌介导胃黏膜恶性转化中的作用及机制。

本课题通过circRNA测序分析获得了幽门螺杆菌感染后胃癌细胞的circRNA表达谱，并筛选出幽门螺杆菌感染后明显上调的环状RNA circMAN1A2，通过一系列实验探讨了circMAN1A2在幽门螺杆菌诱导的胃癌发生中的作用及分子机制。Hp感染胃癌细胞能够显著上调circMAN1A2的表达，且这种上调作用具有时间依赖性，但不依赖于Hp毒力因子CagA，而活菌与细胞的作用对于上调circMAN1A2的表达是必须的。下调circMAN1A2可抑制胃癌细胞的增殖、迁移和侵袭。CircMAN1A2可通过海绵机制吸附miR-1236-3p，干扰circMAN1A2后，miR-1236-3p靶基因MTA2表达下调。在胃癌细胞AGS和BGC-823中感染Hp后，MTA2表达上调。双荧光素酶报告基因检测结果证实了circMAN1A2与miR-1236-3p存在相互结合。qRT-PCR结果表明，在胃癌组织标本中circMAN1A2与MTA2的表达呈正相关。因此，circMAN1A2可通过海绵机制吸附miR-1236-3p调控MTA2的表达，促进幽门螺杆菌诱导的胃癌进展。此外，在裸鼠体内，circMAN1A2的下调抑制了异种移植瘤的生长。在人体组织样本中circMAN1A2的高表达与胃癌的进展有关。因此，幽门螺杆菌通过调控circMAN1A2表达促进胃癌的发生，circMAN1A2可能作为一种新的潜在的胃癌诊断标志物和治疗靶点。