| Esophageal cancer is regarded as the ninth most common malignancy worldwide. Compared with other nations, there is higher incidence in China. Esophageal cancer is an extremely fatal disease. In spite of major advancement in cancer treatment, prognosis is still poor.Owing to recent advancements in tumor biology, apoptosis plays an important role in organ homeostasis, impairment of apoptosis facilitates the accumulation of gene mutations by prolonging the cell cycle span and promoting resistance to immune-based cytotoxicity, finally contributing to carcinogenesis. Survivin is an inhibitor of apoptosis protein, which blocks apoptosis induced by a variety of triggers. Survivin plays an important role in the development and progression of cancers. The expression of survivin is ubiquitous in fetal tissues, but is negligible in the majority of terminally differentiated adult tissues. In various cancers including esophageal cancer, survivin is significantly overexpressed. However, mechanism of survivin being re-expressed in cancers is still unclear and remains to be further studied.Many researches using RT-PCR showed a high level of survivin mRNA in cancer tissues compared with normal tissues, indicating that the survivin overexpression in cancers may arise predominately at the level of transcriptional regulation of the survivin gene. There are many transcription factors regulated survivin transcription in various cell types, including Sp1, Sp3, Egr-1, Stat3Rb, E2F and p53. Among these transcription factors, p53 is especially important in tumors. Like survivin, p53 is also a critical mediator of apoptosis and tumorigenesis. The p53 gene is mutated in various human cancers. In vitro transient transfection demonstrated that the expression of wild type p53 was associated with strong repression of the survivin promoter activity in various cell types, while mutant p53 wasn't. Several clinical studies revealed that the p53 gene mutation might contribute to aberrant survivin expression in non-small cell lung cancers, breast cancers and laryngeal squamous cell carcinomas. While other clinical studies displayed different results. Kawasaki et al indicated that the survivin expression in colon cancer tissues had no relation with p53 mutation; Tanaka et al drawed the same conclusion in breast cancers. Whether the p53 gene mutation contributes to aberrant survivin expression in esophageal cancers or not is yet to be determined. In order to study the possible correlation between p53 and survivin in esophageal cancers, we compared survivin expression between the cancer tissues of wild type p53 and that of mutant p53.Single-nucleotide polymorphisms (SNP) are inheritable mutation and had steady correlation with various diseases. Several polymorphisms in survivin promoter have been identified previously. These SNPs might be correlated with the risk of tumors. The SNPs data of survivin gene in the NCBI (National Center for Biotechnology Information) SNP database are mainly submitted by European, American or Japanese, and the majority of SNPs have no information about the frequency distribution in different race. The SNP frequencies in various race are different, the SNPs'distribution in survivin gene region in Chinese Han nationality and the function of these SNPs have not been satisfactorily described yet. After searched the public SNP database (http://pga.gs.washington.edu, http://www.ncbi.nlm.nih.gov) and literatures, we identified five SNPs, -31G/C, -141G/C, -241T/C, -644T/C and -625G/C. In the present study, we compared these SNPs'distribution in Chinese Han nationality of Chongqing city with the frequency distribution in other race of people. Then we analyzed the relationship between the survivin promoter polymorphisms and the survivin expression of corresponding tumor tissues in order to detect whether the SNPs influence survivin expression or not. Moreover, we have compared the frequency of four survivin promoter polymorphisms in Chinese sporadic esophageal cancer patients and healthy controls in an attempt to identify a possible association between individual genetic variation and susceptibility to esophageal cancer.CpG methylation is thought to be critical for regulating transcription by the GC rich region around the transcription start site. Survivin has a large CpG island spanning from promoter to exon1 region, which increases the possibility that survivin overexpression in cancers may be the result of the dys-methylation status in neoplastic cells. However, Li and Altieri demonstrated that the CpG island in the survivin promoter was unmethylated in both normal and neoplastic tissues in breast, lung and colon cancers. In contrast, there were other researches reporting that demethylation of CpG sites in the survivin gene exon 1 was involved in the survivin expression in ovarian and oral cancers. These different data indicated that the methylation status of the human survivin promoter might depend on the different CpG sites reserchers detected. We are interested in the methylation status of survivin exon 1 region in esophageal cancer, so whether the CpG island in survivin gene is methylated in normal cells and unmethylated in esophageal cancer were studied in this research.In summary, our study is to investigate the mechanisms of survivin overexpression in esophageal cancer, the following data showed the contents and results of our study:1. Esophageal cancer with mutant p53 showed higher survivin gene expression. We measured survivin mRNA expression using RT-PCR in esophageal cancer and adjacent non-cancerous tissues of 100 patients. PCR-single-strand conformation polymorphism (SSCP) analysis was used to detect exons 5–8 mutations of the p53 gene in espohageal cancer tissues. To determine whether the loss of wild type p53 enhanced survivin gene expression in esophageal cancer tissue, the relationship between survivin mRNA expression and p53 gene mutation was analyzed. The ratio of survivin-positive tumors was significantly higher in tumors with mutant p53 than that in tumors with wild type p53 (P=0.020).2. Polymorphisms of survivin promoter were identified using the PCR-RFLP technique (-31G/C, -141G/C, -241T/C) or primer-introduced restriction analysis-PCR assay (-644T/C, -625G/C). We compared these SNPs'distribution in Chinese Han nationality of Chongqing city with the data in NCBI database about the frequency distribution in other race of people. We found significant differences in allele frequencies of -31G/C, -241T/C and -644T/C polymorphisms between Chinese Han nationality of Chongqing city and the NCBI data of global people. The allele frequencies of -31G/C and -644T/C polymorphisms in Chinese Han nationality of Chongqing city were equal to the NCBI'Asia data, but were different to the European and African data. There was significant difference in allele frequencies of -625G/C polymorphisms between Chinese Han nationality of Chongqing city and the NCBI data of Asia and African people.3. Polymorphisms of Survivin Promoter Are Associated with Risk of Esophageal Cancer. The frequencies of -644T/C and -31G/C polymorphisms were not significantly different between patients and controls. However, the significant differences in both allele and genotype frequencies between esophageal cancer patients and cancer-free controls were found in -625G/C polymorphism. Compared with the other genotype, the -625CC genotype was associated with significant elevated risk of esophageal cancer (OR=2.120, 95%CI: 1.247-3.603).4. We examined the combined effect of the survivin promoter polymorphisms, the haplotypes constructed of -644T/C, -625G/C, -31G/C revealed significant associations with esophageal cancer (global P = 0.0034). -644T--625C--31C was a risk haplotype for esophageal cancer (P=0.0001) and -644T?-625G?-31C, a protective haplotype (P = 0.0165).5. Survivin expression was associated with survivin promoter polymorphisms. The difference between the groups of different survivin expression level was statistically significant to the -625G/C polymorphism (P = 0.024), but not obvious for the -31G/C and -644T/C polymorphisms. The variant C-allele in the -625G/C SNP site was associated with a higher possibility of survivin expression in esophageal cancer patients. Furthermore, the patients with survivin-negative cancer tissues were more likely to exhibit the GG genotype (p=0.033).6. We investigated the methylation profiles of the survivin exon1 using BSP method in esophageal cancer and adjacent non-cancerous tissues of 4 patients. The cancer tissues we studied were survivin-positive and matched normal tissues were survivin-negative. After BSP, we didn't find any cytosines in the PCR products'sequences. Therefore, the survivin exon 1 had no methylated CpG site. The survivin-negative non-cancerous tissues and the survivin-positive cancer tissues didn't show different methylation status in the regions at least in the samples we studied.Our results revealed an association between p53 mutation and increased survivin expression in esophageal cancer. At the same time, we found the survivin promoter -625CC genotype was more likely to have survivin expression in cancer tissue. These provided substantial mechanism contributing to survivin up-regulation in esophageal cancer. Furthermore, our case-control study suggests a significant association between survivin promoter SNP and esophageal cancer. Moreover, we identified the high consistency of a common risk (-644T?-625C?-31C) and a protective (-644T?-625G?-31C) haplotype in survivin promoter linked with esophageal cancer. To our knowledge, the present study is the first report demonstrating that survivin expression is negatively regulated by p53 gene status in esophageal cancer patients. This is also the first research showing a significant relationship between the survivin promoter -625G/C polymorphism with its mRNA expression and the risk of esophageal cancer. Although the association was statistically significant with the small number of samples, this needs to be confirmed with a larger sample.
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