FOXM1调控GRP78的基因转录参与胃癌转移的分子机制研究

Endoplasmic reticulum stress (ERS) plays a crucial role in the development of gastric cancer. A growing body of research shows the relationship between ERS and tumor metastasis is increasingly close. Glucose regulated protein 78 (GRP78) is the key protein of endoplasmic reticulum stress. Our previous study had shown that the expression level of GRP78 mRNA in gastric carcinoma were significantly higher than that in chronic atrophic gastritis and superficial gastritis. GRP78 expression was positively correlated with infiltration depth, differentiation, disease stage, lymphoid node metastasis. Our early research found that FOXM1 as an important transcription factor can regulated the transcription of GRP78. Therefore, we hypothesize that FOXM1 involve in gastric carcinogenesis by regulating GRP78 expression. In this project, RNA interference, overexpression vectors and relevant molecular biological techniques would be applied to explore the molecular mechanisms underlying FOXM1-mediated activation and regulation of GRP78 expression. Furthermore,using nude mice models, we plan to determine the functions of FOXM1-GRP78 signaling and underlying mechanisms in gastric carcinogenesis metastasis. This project aims to investigate the biological mechanisms of FOXM1-GRP78 signal pathway in gastric cancer and uncover experimental evidence for its utility for early diagnosis, prevention and treatment of gastric cancer.

内质网应激（ERS）在胃癌发生发展过程中发挥重要作用。越来越多的研究显示ERS与肿瘤转移的关系日益密切。GRP78是内质网应激诱导表达的关键蛋白。我们的前期研究显示GRP78在胃腺癌组织中表达显著高于胃炎组织，其表达与胃癌的分化程度、淋巴结转移、病理分期、浸润深度及肿瘤复发相关。课题组前期实验发现FOXM1作为一种重要的转录因子对GRP78的转录具有调节作用。在此基础上，本课题组提出FOXM1调控GRP78转录参与胃癌转移的分子机制假说。本课题组拟以胃癌细胞株为模型，使用RNA干扰、过表达载体及相关分子生物学技术，探讨FOXM1对GRP78转录激活的调节及其分子机制，最后在裸鼠体内注射胃癌细胞株，观察FOXM1对GRP78转录激活的调节对肿瘤形成、转移的影响。通过研究以期揭示FOXM1-GRP78信号通路在胃癌转移中的分子机制，为肿瘤的早期诊断、预防和治疗等临床工作的开展提供实验室依据。

内质网应激在胃癌发生发展过程中发挥重要作用。糖调节蛋白78（glucose-regulated protein of 78kDa，GRP78）是内质网应激诱导表达的关键蛋白之一。前期研究显示GRP78在胃腺癌组织中表达显著高于胃炎组织，其表达与胃癌的病理分期、淋巴结转移、浸润深度及化疗敏感性、肿瘤复发等临床病理资料相关。并且发现转录因子内质网应激在胃癌发生发展过程中发挥重要作用。糖调节蛋白78（glucose-regulated protein of 78kDa，GRP78）是内质网应激诱导表达的关键蛋白之一。前期研究显示GRP78在胃腺癌组织中表达显著高于胃炎组织，并且发现转录因子FOXM1对GRP78的转录具有调节作用。本课题通过研究以期揭示GRP78的调节蛋白FOXM1对其调控机制，及二者在胃癌转移中的意义。本项目研究结果：GRP78的表达与浸润深度、分化程度、淋巴结转移、临床分期、脉管癌栓有关，Ki-67与浸润深度、分化程度、淋巴结转移、临床分期有关。GRP78与Ki-67的表达呈正相关（r=0.5025，P<0.05）。GRP78及Ki67共表达阳性患者的总生存期低于共表达阴性者，差异有统计学意义。GRP78表达阳性患者使用两药及以上联合化疗方案对比氟尿嘧啶类单药方案总生存时间的差异有统计学意义。qRT-PCR和免疫印迹方法显示过表达FOXM1后，GRP78在蛋白及mRNA表达水平均增加。荧光素酶报告基因实验结果显示FOXM1过表达组荧光素酶活性高于对照组。ChIP和EMSA结果显示FOXM1能够与GRP78启动子区域结合并发挥调控作用。qRT-PCR方法和免疫印迹显示不同胃癌细胞中FOXM1的蛋白和mRNA表达水平，MKN45表达最低，MKN28表达最高。qRT-PCR结果显示衣霉素诱导胃癌细胞发生内质网应激后应激蛋白表达高于对照组。免疫印迹方法结果显示过表达FOXM1组应激相关蛋白的表达也明显高于对照组。Transwell小室实验显示FOXM1过表达组小室下层细胞数目明显多于对照组。免疫共沉淀实验显示胃癌细胞中GRP78和KIAA1199能够结合，细胞荧光显示二者存在共定位。FOXM1调控GRP78能够促进胃癌细胞的迁移等能力，提示二者有望成为胃癌的潜在临床检测指标和治疗靶点，为肿瘤的早期诊断、预防和治疗等临床工作的开展提供实验室依据。