PLCE1基因调控炎性NF-κB信号通路在新疆哈族食管癌侵袭转移中的作用与机制研究

The Kazakh population, in Xinjiang, is characterized by having higher incidence of esophageal carcinoma than those in the general population of China. The most important factor affecting the prognosis of a person with esophageal carcinoma is the migration and invasion. However, the inflammatory tumor microenvironment palys an important role in promoting the migration and invasion of esophageal carcinoma. It has been reported that the NF-κB, an inflammation-associated signal pathway, is offen activated in the esophageal carcinoma, whereas the exact mechanisms on how regulate the NF-κB is not clear. In our previous research, we have found that the up-regulation of PLCE1 expression can promote the invasion and migration of Kazakh esophageal carcinoma, which is accompanied by the notable characteristic of epithelial-mesenchymal transition (EMT). We also found that the overexpression of PLCE1 is positively associated with the NF-κB signal pathway protein, i.e.,IKK and p65. Therefore, we would hypothesize that PLCE1 gene could cooperate with NF-κB to form the inflammatory tumor microenvironment and lead to the development of EMT. To verify the hypothesize, firstly, we will analyze the relationship of the expression of PLCE1 and NF-κB in the phenotype transformation of tumor cell in esophageal carcinoma tissues. Secondly, using knockdown of PLCE1 expression by shRNA and inducing overexpression of it, we will research the influence of different expression level of PLCE1 on the EMT induced by the inflammatory tumor microenvironment in esophageal carcinoma cell lines and clarify the molecular mechanisms of the PLCE1regulate the transcriptional activity of the NF-κB signal pathway. Finally, we will investigate the therapeutic action of inhibiting the NF-κB signal pathway regulated by the PCLE1gene via forming the model of inflammatory microenvironment of esophageal carcinoma in vivo. Depending on all above studies and experiments, we hope find the new mechanisms of regulating the NF-κB signal pathway in invasion and migration of Kazakh esophageal carcinoma.

新疆哈族食管癌发病率远高于全国平均水平。侵袭转移是影响预后的重要因素，而炎性微环境对食管癌侵袭转移具有重要作用。炎症相关NF-κB通路在食管癌中常呈异常活化，但其促进侵袭转移机制了解甚少。我们前期研究发现PLCE1基因上调可促进哈族食管癌侵袭转移并伴显著EMT过程，且蛋白高表达与其下游NF-κB通路相关分子呈正相关。由此我们推测PLCE1可能通过调控NF-κB促进食管癌炎性微环境形成和EMT发生。为验证此假说，本项目拟首先在哈族食管癌组织中分析PLCE1与NF-κB表达及肿瘤细胞表型转化的关系；其次，通过诱导和阻断PLCE1表达，研究其对炎性微环境诱导的食管癌细胞EMT的影响，并探讨PLCE1调节NF-κB转录活性的分子机制；最后，构建食管癌炎性微环境在体模型，阐明干预PLCE1/NF-κB通路对食管癌侵袭转移的治疗作用，以期从干预炎性微环境角度防治哈族食管癌的侵袭转移提供新的靶点和方向。

新疆哈族食管癌发病率远高于全国平均水平，侵袭转移是影响预后的重要因素，而炎性微环境对食管癌侵袭转移具有重要作用。炎症NF-κB通路在食管癌中常呈异常活化，根据前期研究我们推测PLCE1可能通过调控NF-κB促进食管癌炎性微环境形成和EMT发生。通过本项目的实施，我们发现，1. PLCE1、PKCα、IKKβ、IKBα、p50及p65蛋白在新疆食管癌组织中高表达，PLCE1与p65，IκBα，IKKα/β的表达具有显著正相关性。PLCE1、IKBα、p53高表达的ESCC患者术后生存率显著低于低表达组，联合PLCE1、IKBα、p53蛋白对ESCC患者术后预后较差具有较高的预测价值；2. PLCE1基因被抑制后MTT和单克隆形成实验显示食管鳞癌细胞的生长受到明显抑制，与对照组相比，早期和晚期凋亡的比率明显升高，食管鳞癌细胞株中凋亡相关蛋白和的表达量明显增高； PLCE1基因被抑制后，MTT实验显示人脐静脉内皮细胞的生长受到明显抑制，侵袭以及成小管能力显著降低，血管生成相关分子VEGF-C的表达显著降低；3. PLCE1可以直接与p65、IκBa分子相结合，同时促进p-IκBα（Ser36）、IKKα/β（Ser176/180）、p65（Ser536）的磷酸化，并促进p65核内转移，从而激活NF-κB通路，转录因子p65分子可以与VEGF-C-164~-62段启动子相结合，还可以作用于Bcl-2启动子的-1423~-1386段；PLCE1基因以及NF-κB路被同时抑制后，MTT显示食管鳞癌细胞的生长受到显著抑制，与对照组相比，早期和晚期凋亡的比率显著升高，食管鳞癌细胞株中凋亡相关蛋白和的表达量显著增高；4. PLCE1基因以及NF-κB通路被同时抑制后，MTT实验显示人脐静脉内皮细胞的生长受到明显抑制，侵袭以及成管能力显著降低，血管生成相关分子VEGF-C的表达显著降低；5.食管鳞癌细胞系Eca109细胞裸鼠移植瘤模型中，PLCE1基因以及NF-κB通路被同时抑制时，裸鼠成瘤体积与瘤重量显著降低，血管生成相关蛋白表达显著降低，凋亡相关蛋白和的表达量显著增高。对易感基因PLCE1参与食管炎-癌恶性转化中的分子机制的深入研究，可为新疆地区食管鳞癌高危人群预警及个性化治疗提供新的思路和潜在的靶点。