TMEFF2对胃癌生物学行为的影响及其与STAT3相互作用的机制

Gastric cancer is a major cause of cancer-related death worldwide. The pathogenesis and the target in the prevention of gastric cancer are the research focus lately. Many researches found that the abnormal regulation of many genes and signaling pathway related with the development of gastric cancer. However,the biological and molecular mechanisms underlying gastric cancer development remain largely unclear. Recently, we found the abnormalities expression of signal transducer and activator of transcription3 (STAT3) in gastric cancer. The promoter array indicated that STAT3 may bind to the transmembrane protein with EGF-like and two follistatin-like domains 2 (TMEFF2) promoter. TMEFF2 was expressed in normal human gastric epithelial cell line (GES1).However, it was silenced or downregulated in 4 out of 5 gastric cancer cell lines. Restored expression of TMEFF2 dramatically suppressed cell growth curve in gastric cancer cell lines. We also found that STAT3 could regulate the expression of TMEFF2. TMEFF2 could also in turn regulate STAT3 expression. Therefore, the interaction of TMEFF2 with STAT3 may play an important role in gastric cancer. However, the effect of TMEFF2 on the biological behavior of gastric cancer and the mechanism of interaction of TMEFF2 with STAT3 are poorly understood. We aim to clarify the expression of TMEFF2 in the clinical significance of gastric cancer, to study the biological function of TMEFF2 and to explore the mechanism of the interaction of between TMEFF2 and STAT3 in gastric cancer. These findings may provide new insights into the molecular mechanisms of gastric cancer pathogenesis and may lead to new approaches for the early detection and effective therapy of gastric cancer.

胃癌发生机制及防治靶点一直是研究热点。研究发现胃癌的发生涉及基因调控失常和信号通路传导异常，但具体机制不明。最近我们发现在肿瘤中发挥重要作用的信号转导及转录激活因子3(STAT3)在胃癌组织中异常激活；启动子芯片显示STAT3可能结合TMEFF2启动子区；TMEFF2在正常胃组织中高表达，在胃癌中表达下调；过表达TMEFF2可抑制胃癌细胞增殖。据此我们推测TMEFF2作为STAT3的靶基因在胃癌中发挥重要作用，但STAT3信号通路调控TMEFF2表达的分子机制以及其相互作用的具体机理罕有研究。因此，本研究拟在前期工作的基础上，通过组织标本、细胞学实验和动物体内实验：①探讨TMEFF2表达与胃癌患者临床病理特征的联系及与预后的关系②明确TMEFF2对胃癌生物学行为的影响③研究TMEFF2与STAT3相互作用的机制。以揭示STAT3-TMEFF2通路在胃癌发病中的意义，为胃癌治疗提供理论依据。

胃癌致死率在世界范围内位居恶性肿瘤第二位，胃癌的发生发展涉及多种基因的调控异常和信号通路的传导异常。采用基因表达谱芯片研究胃癌及其配对的癌旁组织中表达差异的基因发现TMEFF2在70%胃癌组织中表达降低显著。TMEFF2是I型进化保守的跨膜蛋白，由一个表皮生长因子类似结构域和两个卵泡抑素类似结构域组成。荧光定量PCR和免疫组化结果显示从胃正常黏膜经萎缩伴肠化、异型增生到肠型胃癌的过程中 TMEFF2 的表达逐渐降低。TMEFF2 在44.6%的胃正常黏膜组织高表达, 在29.2%萎缩伴肠化组织中高表达。仅有18.5%的异型增生组织TMEFF2 高表达, 而胃癌组织中仅有7.7%的标本为TMEFF2 高表达。生存分析发现TMEFF2高表达的胃癌患者预后较好(P=0.0148; HR, 0.57; 95% CI, 0.37–0.90)。体内与体外实验均发现TMEFF2可以抑制胃癌细胞增殖、使胃癌细胞发生G2/M期阻滞、促进胃癌细胞凋亡。TMEFF2 表达下调可以引起 DNA 损伤、增加基因组不稳定和 DNA 突变频率。我们利用基于质谱分析的蛋白质组学方法发现SHP-1可能与TMEFF2相互作用。Co-IP证实了两者存在相互作用，免疫荧光显示存在共定位。生物信息学分析结果显示，TMEFF2蛋白含有3个结构域，而SHP-1蛋白含有2个SH2结构域和1个PTPD结构域。截断突变实验显示，TMEFF2ID（TEMFF2细胞内结构域） 对于TMEFF2/SHP-1相互作用和TMEFF2在胃癌中的功能非常重要。另一方面，在胃癌细胞中SHP-1可介导TMEFF2的生物学行为如增殖和凋亡等。我们进一步分析人体胃组织不同病理阶段SHP-1与TMEFF2 表达的相关性。结果显示从胃正常黏膜到肠型胃癌SHP-1和 TMEFF2 的表达显著负相关。此项研究揭示了TMEFF2在胃癌中的生物学功能、临床意义及与SHP-1相互作用的机制，可能为胃癌的早期预警和胃癌的治疗提供新的靶点。