【作者】 付涛；

【导师】 刘宝华；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 EPHB2基因编码的蛋白EphB2是受体酪氨酸激酶家族的一员,表达于细胞膜,与相邻细胞表面的配体结合介导双向信号转导,在膜蛋白酶水解作用或内吞作用下受体、配体之间的结合被破坏,从而导致细胞间的排斥。早期研究发现EphB2在人类多数肿瘤(包括结直肠癌)中存在表达上调,能通过提升细胞迁移能力和促进血管生成,参与肿瘤的形成和发展。但新近研究却发现EphB2在结直肠腺瘤-癌过渡时期存在不同程度的表达下调,它与结直肠癌的发生、发展密切相关,主要通过与其配体结合介导细胞间排斥(区室化)抑制结直肠上皮细胞癌变及进展。结直肠癌中EphB2表达下调的原因还不清楚。目前,仅发现极少数结直肠癌中存在EPHB2基因的突变、杂合性丢失等遗传学改变。而关于表观遗传学改变也存在一定争议。因此有必要对结直肠癌中EphB2的表达和调控进行进一步的研究。在本研究中,我们采用Western印迹法检测了4株结肠癌细胞EphB2的表达,采用免疫组织化学和Real-time PCR技术检测了13例正常人直肠粘膜组织、30例结直肠癌及癌旁正常肠粘膜组织中EphB2蛋白和mRNA表达水平,证实结直肠癌中EphB2的表达存在不同程度的下调,其表达水平与肿瘤分化程度有关(P<0.05),分化越差,表达越低。采用甲基化特异性PCR技术检测了上述细胞和标本中EPHB2基因启动子区CpG岛甲基化状态,结果发现无一例存在甲基化。同时采用亚硫酸氢盐修饰后基因组测序技术在所有细胞和其中17例EphB2缺失表达的结直肠癌组织中验证了以上结果。由于转录调控是基因表达调节的重要环节,目前关于EPHB2这方面的研究报道较少。我们首先采用报告基因和荧光素酶检测系统对EPHB2基因启动子进行克隆、鉴定,确定核心启动子位于-425~+83区域内,并发现-1174~-970区域存在沉默子。生物信息学分析发现这两个区域分别存在转录因子Sp1的结合位点之一GC盒和转录因子c-Rel的结合位点。我们对这两个位点进行突变后发现,GC盒突变后启动子活性较突变前降低了15%左右(P>0.05);c-Rel结合位点突变后启动子活性较突变前升高了85%左右(P<0.01)。综上所述,结直肠癌中EphB2的表达无论在蛋白还是mRNA水平均存在不同程度的下调,其原因与基因启动子区CpG岛甲基化无关,而与转录调控有关。在结直肠癌中,可能是转录因子c-Rel活化后与沉默子结合,从而抑制了EPHB2基因的转录表达。以上的研究结果为阐明结直肠癌中EphB2表达下调机制提供了新的线索,也有希望为结直肠癌的基因和药物治疗提供新的靶点。更多还原

【Abstract】 EphB2 receptor coded by EPHB2 gene is a member of receptor tyrosine kinase. They are located on the cell surface, and transduce bi-directional signals when binding with their ligands on adjacent cell surface. Being cleaved by transmembrane proteases or removed from the cell surface by trans-endocytosis results in cell-cell repulsion. EphB2 was found at high levels in diverse human tumor types, including colorectal cancer (CRC). Elevated expression has been proposed to promote tumorigenesis by modulating cell motility and angiogenesis. But recent studies have found most human CRCs lose expression of EphB2 at the adenoma-carcinoma transition. It represents a critical step in CRC progression. EphB2-mediated compartmentalization by binding with its ligand restricts the spreading of tumor cells.The mechanisms of EphB2 expression silencing in CRC remain uncharacterized. Genetic changes, such as point mutations and loss of heterozygosity, were found in a fraction of CRCs. There are conflicting data regarding epigenetic regulation of EPHB2 in CRCs. Anyway, a further research on the expression and its regulation is helpful.In this study, we examined the expression of EphB2 in four colon cancer cells, 13 normal rectal mucosas, 30 CRCs and matched normal colorectal mucosa samples by using western blot, immunohistochemistry or real-time PCR. We found that expression of EphB2 was silenced in most of the CRCs. A significant association (P<0.05) was found between EphB2 expression and histological differentiation. Tumors with negative EphB2 expression were more often poorly differentiated. Methylation status of promoter CpG island of EPHB2 in all tissues and 4 colon cancer cells were determined by methylation specific PCR, and showed a complete absence of methylation. These results were confirmed in 17 CRCs with negative expression of EphB2 and the four colon cancer cells by bisulfite genomic sequencing.For the importance of transcriptional regulation in gene expression but the lack of detailed experimental data in the human EPHB2 promoter, it promoted us to not only investigate the responsible regions in the 5’flanking region of the human EPHB2, but also the transcription factors associated with the human EPHB2 transcript expression. Transfection assay using a series of deletion constructs of the 5’-flanking region fused to the luciferase reporter gene revealed that a 508-bp (-425/+83) fragment are necessary for high transcriptional activity, and identified a 205-bp (-1174/-970) fragment with silencer activity. The computer analysis predicted the presence of a cis-element (GC-box) in promoter region for Sp1 and a cis-element in silencer region for c-Rel. Mutation of GC-box resulted in a decline of about 15% in EPHB2 promoter activity compared to wild type promoter activity (P>0.05). Mutation of c-Rel binding site resulted in a highly significant increase of about 85% in EPHB2 promoter activity compared to wild type promoter activity (P<0.01).Our data indicated that expression of EphB2 was silenced at different degree in most CRCs. The silencing occured at both transcriptional and protein levels, and was not associated with methylati4on of EPHB2 promoter CpG island, but with transcriptional regulation. We postulated that transcription of EPHB2 gene is regulated negatively by binding c-Rel transcription factor on the silencer, which results in the silencing of EphB2 in CRCs. These results provide a new clew to investigate the mechanisms of EphB2 silencing in CRC, as well as a new target for gene therapy and drug treatment of CRCs.更多还原