CREB1/CRTC2复合物调控Snail表达诱导EMT促进胃癌侵袭转移的分子机制研究

Gastric cancer (GC) is one of the malignant tumors with high morbidity and poor prognosis. Tumor invasion and metastasis is the main cause of GC patients’ death. Unfortunately, the molecular mechanism is not clear yet. Our preliminary experiments showed that inflammatory factor IL-1β could mediate epithelial-mesenchymal transition (EMT) to enhance the migration and invasion of GC cells. Meanwhile, the expression of the transcription factor CREB1 and its co-activator CRTC2 in GC samples were both upregulated and correlated with chronic inflammation. CREB1 could promote migration and invasion of GC cells and was activated by PI3K/Akt signaling pathway. There are CREB1 binding sites in the promoter region of Snail, a key transcription factor of EMT. Therefore, the scientific hypothesis of this proposal is that inflammatory factor IL-1β could mediate EMT to promote invasion and metastasis of GC through CREB1/CRTC2 complex. We will elucidate the whole process that inflammatory factor IL-1β activates PI3K/Akt signaling pathway, cause transcription factor CREB1 translocating into the nucleus after phosphorylation, form complex with its co-activator CRTC2, recruit CBP/P300 for histone acetylation, promote downstream target gene Snail transcription, and mediate EMT to enhance GC cell invasion and metastasis. This study will help to deepen the molecular mechanism of invasion and metastasis of GC and provide a theoretical basis for the prevention and treatment of GC.

胃癌发病率高、预后差，肿瘤侵袭转移是患者死亡的主要原因，但其分子机制还不清楚。我们前期研究发现，炎症因子IL-1β能介导EMT增强胃癌细胞迁移侵袭能力；胃癌临床样本中转录因子CREB1和其共激活因子CRTC2表达均明显上调，并与慢性炎症相关；CREB1可促进胃癌细胞迁移侵袭，并受到PI3K/Akt信号通路的活化；分析EMT关键转录因子Snail基因启动子结构发现存在CREB1的结合位点。因此，本项目提出IL-1β通过CREB1/CRTC2转录复合物激活Snail介导EMT促进胃癌侵袭转移的科学假说，将阐明炎症因子IL-1β通过激活PI3K/Akt信号通路，使CREB1发生磷酸化入核，与CRTC2形成复合物，并招募CBP/p300进行组蛋白乙酰化修饰，激活Snail转录，介导EMT相关基因表达，促进胃癌转移的作用机制。本研究将有助于完善胃癌侵袭转移分子机制，为胃癌防治提供理论基础。

我国胃癌的早期诊断率低，多数患者就诊时已发生侵袭转移，因而造成治疗失败，甚至患者死亡，但目前对胃癌发生侵袭转移的分子机制尚不清楚。因此，深入研究胃癌侵袭转移的机制将为其防治提供必要的理论支持。课题组前期用转录组芯片研究了111例胃癌组织和21例正常胃粘膜组织基因表达谱，以差异共表达网络分析发现，与正常对照相比，转录因子与下游基因的调控关系在肿瘤组织中发生了明显改变，其中CREB1就是一个核心的转录因子，其表达差异高达12.85倍。进一步通过TCGA-STAD、GSE54129及收集的胃癌组织进行CREB1的表达和临床意义分析，对其差异表达基因进行KO、KEGG、GSEA分析，并通过cox回归分析构建列线图模型，以评价CREB1在胃癌中的临床意义及作为独立预后指标的能力。干扰胃癌细胞CREB1的表达后，采用细胞增殖、平板克隆及流式分析技术检测下调CREB1对胃癌细胞增殖、凋亡及细胞周期的影响；采用Transwell和划痕愈合实验检测下调CREB1对胃癌细胞迁移能力的影响；建立裸鼠皮下瘤、腹腔种植和肺转移模型，检测下调CREB1对胃癌细胞在体成瘤、腹腔播散以及肺转移能力的影响。在慢性炎症微环境下，通过检测CCK-8、流式检测、Transwell实验及real-time PCR检测炎症微环境对胃癌细胞增殖、凋亡及迁移侵袭的影响。IL-1β通过激活PI3K/Akt上调CREB1表达，荧光素酶报告基因验证CREB1直接调控Snail，促进胃癌侵袭转移。本研究揭示CREB1在胃癌发生发展中具有重要作用，阐明炎症因子IL-1β上调CREB1促进胃癌侵袭转移的分子机制，初步探索CREB1作为治疗靶标在临床应用中的可能性和策略。