慢性胃炎向胃癌转化的转录调控网络及其关键节点的研究

Epidemiological studies provide associations between chronic gastric inflammation and gastric cancer; however, the mechanisms are largely unknown. The molecular regulators and transcriptional networks during the progresses of gastritis to gastric cancer are not documented yet. The goal of this project is to investigate the progress of chronic inflammation to carcinogenesis in stomach by using clinical samples from patients and healthy volunteers. The RNA and DNA database of normal gastric tissues, precancerous lesions and gastric cancer tissues are constructed by using cDNA microarray and aCGH analyses. The differently expressed genes within different stages of disease are obtained by large-scale comparing based on computing platform. The key regulators and its cohort are identified using biosystematic methods including master regulator analysis, stochastic dynamics and Bayesian probability analysis, etc. Large-scale perturbation cell models are used to rank the interactions between regulators and its target genes. The transcriptional networks are thus constructed and separated by functional modules. Gastric cancer cell lines are used to validate the roles of key regulators and deepen the signaling pathway investigations. Nude mice models are used for in vivo analysis. These findings provide understanding of mechanisms about chronic gastric inflammation progresses to gastric cancer; furthermore, propose promising targets for clinical intervention.

流行病学证据支持慢性胃炎发展为胃癌，但确切机制尚不完全清楚，从整体上理解慢性胃炎向胃癌转化过程中分子调控机制及其调控网络研究尚无报道，本项目拟以临床病人样品为出发点，以正常组织、慢性萎缩性胃炎、胃癌的病理发展过程为研究对象，运用cDNA microarray和aCGH技术，分别构建正常组织、癌前病变组织和胃癌组织RNA表达谱和DNA拷贝数数据库。通过大规模数据比对，找出在差别表达数据，结合转录因子活性分析、随机动力学和贝叶斯概率分析等方法，建立慢性炎症向胃癌的转录调控网路并揭示其关键节点。运用大规模扰动加以确认，并以胃癌细胞株和动物模型加以验，进一步揭示胃癌发生发展的分子机制。

我们应用差异共表达分析策略，根据基因表达谱信息筛选出富集了差异调控关系的差异共表达基因，以这些基因为核心构建条件特异、并富集了差异调控关系的调控子网络。对胃癌和正常条件下的调控网络进行了拓扑属性和功能的分析与比较，据此对基因或调控关系排秩，排秩越靠前的基因或调控关系与疾病发生发展越相关。.研究了幽门螺杆菌（HP）相关胃癌抑癌基因异常高甲基化的病因及分子机制。CagA与抑癌基因MGMT启动子高甲基化及DNMT1高表达正相关。CagA通过增强PDK1和AKT相互作用增强其磷酸化水平，磷酸化AKT进而激活NFκB， 活化的NFκB直接调控DNMT1上调其表达进而促进MGMT等抑癌基因启动子高甲基化。幽门螺杆菌CagA通过PDK1/AKT-NF-KB-DNMT1这条通路促进抑癌基因的高甲基化而参与胃癌的发生发展。.sFRP1蛋白在胃癌组织中高表达，在正常胃上皮细胞中无/极弱表达。sFRP1蛋白表达高与淋巴结转移及患者生存期短相关，可作为评判胃癌病人预后的潜在指标。过表达sFRP1可促进胃癌细胞生长、迁移、侵袭能力，并促进胃癌细胞发生EMT转换；使用人全基因组芯片检测sFRP1过表达引起的基因表达改变发现多种增强成瘤能力的基因（RUNX2、ZEB2、DNMT3L、CD44等），同时也诱导与肿瘤基质重建相关基因（TGFβ1、VEGF、SEPP1、ILs等）和免疫逃逸相关基因（HLA-DRA、PD-L2等）的表达。发现TGFβ1/RUNX2信号通路是介导sFRP1下游的重要途径。.MELK在胃癌组织及细胞中高表达，并与临床病理特征相关，可能起癌基因作用；5-FU刺激能上调MELK的表达，MELK可能参与了肿瘤的化疗抵抗；MELK能促进细胞增殖、迁移和侵袭，并能影响细胞骨架形态，可能是胃癌治疗的潜在靶点之一； MELK促进胃癌细胞迁移和侵袭的机制为：MELK促进FAK、paxillin的磷酸化，从而激活下游蛋白；MELK通过增加RhoA的活性，改变细胞骨架，从而调节细胞的运动。.总之，我们基于胃癌患者肿瘤组织、癌旁组织与正常胃组织的基因表达谱数据，构建了特定条件下的基因调控网络，并通过差异分析识别到一批胃癌发生发展相关的关键调控因子，如E2F、PHF10、MELK、sFRP1、RegIV、miR-409-3p和miR-155。我们发现MELK受E2F调控，MELK可以磷酸化FAK和