TGF-β/Smad信号通路在胃癌发展中的角色转换及机制研究

TGF-β plays a dual role in the progression of huaman gastric cancer. During the early stages of cancer, TGF-β function as a tumor suppressor. During the advancing stages of tumor development, TGF-β can promote tumor growth and metastasis. It is still unknown why and how TGF-βevolves into a tumor promoter from a tumor suppressor. Recently, it has reported that a shift in Smad2/3 phosphorylation from the carboxy terminus to linker sites is a key event determining biological function of TGF-β in colorectal and hepatocellular carcinoma. However, the hypothesis has not yet been confirmed in gastric cancer. Our previous studies have presented that their expression of different sites prosphorylation of the Smad protein were different in gastric adenocarcinoma, and indicated that Smad proteins expression was dymatic in gastric cancer. We speculate that this change may be one of reasons of the dual role of TGF-β. In the current study, we plan to investigate the role of p-Smad2/3 either in carboxy terminus or linker sites in gastric cancer. In gastric cancer, immunohistochemistry will be used to detect p-Smad2/3C or p-Smad2/3/L. The correlation between IHC score of p-Smad2/3C/L and characteristics of cancers will be examined. The detailed molecular mechanism of TGF-β/Smad pathway in cancer progression will be investigated in gastric cancer cell lines by using adenoviral and siRNA mediated molecule up/down regulation. Further, we will establish nude mouse gastric cancer model to investigate whether interfering Smad2/3 impact the formation and progression of gastric cancer in the model. In a word,the study will provide detailed mechanisms of TGF-β/Smad pathway in the advancement of gastric cancer and interfering TGF-β/Smad signalling pathway might be a potential therapeutic strategy in the future.

近来linker理论用于解释TGF-β/Smad信号通路早期抗肿瘤和晚期促肿瘤的双向作用机制，但能否适用于胃癌，有待验证。本课题组在前期研究中发现不同位点磷酸化的Smad蛋白在胃癌中表达存在差异，预示Smad蛋白在胃癌发展中可能存在动态变化，该变化可能是TGF-β/Smad信号通路双向作用的原因之一。本课题拟通过检测胃癌不同阶段中Smad蛋白的表达变化，分析其变化规律，明确起主导作用Smad蛋白；通过胃癌细胞株模型，在细胞分子水平上分析TGF-β/Smad通路的网络调控关系，验证Linker理论，揭示Smad蛋白在胃癌细胞中分子机制及双向作用的关键差异基因；建立胃癌移植瘤模型，研究Smad2/3蛋白不同表达对胃癌生长和转移影响，寻求下调胃癌TGF-β/Smad通路中关键靶点。本项目有助阐明TGF-β/Smad通路在胃癌中双向作用的分子机制，为探索一种新的治疗胃癌方法策略提供实验依据。

近来linker理论用于解释TGF-β/Smad信号通路在大肠癌和肝癌中早期抗肿瘤和晚期促肿瘤的双向作用机制，但能否适用于胃癌，有待验证。本课题组通过分析胃癌不同阶段中P-Smad2/3L蛋白和P-Smad2/3C的表达变化，分析其变化规律，明确起主导作用Smad蛋白。在现有的经费支持下，我们观察不同磷酸化Smad2/3在胃腺癌中的潜在作用。使用anti-P-Smad2/3C P-Smad2/3L抗体对130例胃腺癌石蜡标本进行免疫组织化学染色，分析P-Smad2/3C和P-Smad2/3L表达与临床病理的关系。采用实时PCR比较分析胃癌与癌旁组织中Smad2和Smad3的mRNA表达变化。研究结果表明P-Smad2L和/或P-Smad3L在大多数标本中（阳性18/130）没有显著性差异，P-Smad2/3L阳性表达与羧基端磷酸化减少无关。P-Smad2C的缺失明显与肿瘤浸润深度和胃癌细胞恶性分化程度密切相关，P-Smad3C的表达与胃腺癌临床病理特征没有相关。不过，荧光联合染色表明P-Smad3C与α-SMA 和collagen I共同定位于胃癌细胞中，表明P-Smad3C与肿瘤的上皮间质转化存在相关性。实时PCR检测表明，与癌旁组织相比，Smad2- mRNA的在胃癌组织是低表达，与肿瘤的侵犯和胃癌低分化密切相关。与在大肠癌和肝癌组织中相比，经典的羧基末端磷酸化，而不是linker理论，特别是Smad2，在胃癌中起重要的作用。总之，本项目有助阐明TGF-β/Smad通路在胃癌中双向作用的分子机制，寻求下调胃癌TGF-β/Smad通路中关键靶点提供实验依据。