抑癌基因KLF4失活在幽门螺旋杆菌感染致胃癌发生发展的作用及其分子机制

Gastric cancer is one of the most malignant diseases that cause serious harm to human health. The existing data indicate that Helicobacter pylori (HP) infection is the class I bacterial carcinogenic factor for stomach, but little is known about the molecular pathogenesis, especially how HP interacts with host genes to affect the development and progression of gastric cancer. In our previous studies we found: loss or inactivation of KLF4 expression closely correlated with gastric cancer origin, development and prognosis, but the underlying molecular mechanisms leading to KLF4 inactivation in gastric cancer remain unclear. In preliminary experiments we found that: 1) compared with that in normal gastric mucosa, significant promoter methylation and reduced mRNA expression of KLF4 were observed in gastric cancer tissues; 2) HP infection or CagA gene transfection significantly inhibited KLF4 expression. On the basis of these data, we hypothesize that HP infection leads to inactivation of KLF4 tumor suppressor in gastric epithelial/cancer cells through different mechanisms, and thus, promotes the development and progression of gastric cancer. In this project, we plan to use established conditional klf4 gene knockout mouse model, related cell lines, gastric cancer tissue samples and associated clinical data to investigate the causal relationship between HP infection and KLF4 inactivation in the development and progression of gastric cancer, and further elucidate the underlying molecular mechanisms. It is anticipated that the results from this study will provide new insights into and a novel gene target for gastric cancer prevention and treatment.

胃癌是严重危害人类健康的恶性肿瘤之一。已有资料表明，幽门螺杆菌（HP感染是胃癌的第一类生物致癌因子，但对其致病机制及 HP如何与宿主基因相互作用影响胃癌的发生发展知之甚少。本组研究发现：抑癌基因 KLF4 表达的丢失和失活与胃癌的发生发展及预后关系密切，但其在胃癌中失活的分子机制尚不清楚。预实验中发现: 较正常胃粘膜组织，胃癌组织中 KLF4 基因启动子存在明显的甲基化，KLF4 mRNA 表达显著降低;HP 感染或 CagA 基因转染可显著抑制 KLF4 蛋白的表达。我们假设：HP 感染可能通过不同的机制引起KLF4 基因失活，从而促进胃癌的发生发展。本课题拟用已经建立的条件性胃 Klf4基因敲除小鼠模型，相应的细胞系，胃癌组织标本及相关的临床资料，分别从动物，细胞分子生物学及人体水平三个层次观察HP感染致KLF4失活及促胃癌作用的因果关系，并阐明其分子机制，并为胃癌的防治提供新的靶点。

锌指转录因子KLF4在很多肿瘤中起到抑癌基因的作用，在HP感染诱发胃癌发生发展过程中可能导致其表达降低或失活。本课题建立了Villlin-Cre 胃粘膜上皮细胞的 KLF4 基因小鼠模型，以及KLF4敲除细胞模型进行了研究，发现KLF4 基因敲除小鼠胃窦部粘膜呈现过度增生，并见显著的核异型该基因敲除小鼠合并服用化学诱癌剂(N-methyl-N- nitrosourea， NMU)可诱导 Villlin 阳性细胞呈块状扩展所形成的肿瘤细胞内 KLF4 基因丢失。提示数量很少的 VGPC中 KLF4 的失活与肿瘤细胞的起源密切相关，且KLF4敲除细胞其增殖，迁移，克隆能力都是增强的；首先，我们将CagA转染模拟HP感染至胃上皮粘膜细胞显著下调KLF4的表达，提示我们KLF4在胃癌发生发展过程中表达降低或丢失，进一步研究发现CagA转染影响去甲基化蛋白酶TET1的表达从而导致KLF4的表达降低。且在这个过程中CagA诱导胃黏膜上皮细胞炎症因子miR-155的表达上调，可能与EMT的发生有关。另一方面我们又发现，幽门螺杆菌CagA通过去泛素化途径负向调控KLF4的表达。为此，我们建立了幽门螺杆菌细胞的培养方法以及细菌细胞共培养方法，研究HP感染诱导KLF4表达失活的分子机制；同时发现KLF4是miR-155的下游靶基因，CagA通过靶向作用于miR-155下调KLF4的表达，促进了胃黏膜上皮细胞的恶性转化。研究结果为肿瘤发生机制探讨提供指导价值，同时为抑癌基因KLF4在胃癌中表达降低提供可靠证据。通过本课题研究，待发表文章两篇；中文期刊两篇；国内会议两篇；硕士大论文三篇，待毕业一篇；，国家级学术会议大会报告5人次，特邀单位学会报告2人次；三名硕士获得学位。

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