Association Study Between The COX-2 Gene Polymorphism And Head And Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) cancer is one of the most frequent malignant tumors in the world. In recent years, it shows a significantly increasing trend in the last two decades. However, the exact molecular mechanisms to develop head and neck squamous cell carcinoma are still unclear. It is well accepted that interaction of environmental factors and genetic factors may contribute to the etiology of head and neck squamous cell carcinoma. The individuals with different genetic background have different risk of head and neck squamous cell carcinoma. So, it is important to find the head and neck squamous cell carcinoma susceptibility genes in a high-risk population.Overexpressing of cyclooxygenase-2(COX-2) has been implicated in the development of many type of human cancer. Single nucleotide polymorphism (SNP) in the COX-2 gene might contribute to diffenertial COX-2 expressiong and enzymatic activity. The SNP located in the non-coding region, especially in promoter region or 3'untranslated region(3'UTR) of COX-2 may affect the biological functions of COX-2 and risk of developing head and neck squamous cell carcinoma. Thus, we conducted a case-control study to investigate the association of COX-2 genes polymorphisms with head and neck squamous cell carcinoma susceptibility in Chinese populations.Part I: COX-2 gene polymorphism and the susceptibility of head and neck squamous cell carcinomaCOX-2 is the critical enzymes that convert arachidonic acid into prostaglandins and thromboxanes. COX-2 is always involved in inflammation angiogenesis, anti-apoptosis and carcinogenesis. COX-2 overexpression was found in a large proportion of tumor tissues and was significantly associated with advanced tumor stage and lymph node metastasis, such as colon, breast, gland, stomach, prostate and head and neck cancer.The expression of COX-2 is regulated by a complex signal transduction pathway in which many nuclear proteins interact with the COX-2 promoter region and play a decisive role in gene transcription. Therefore, single nucleotide polymorphisms (SNP) in the COX-2 promoter may have a great impact on gene transcriptional activity by altering the binding capability with certain nuclear proteins, resulting in inter-individual variability in susceptibility to cancer. In research of Chinese population, two SNP-1195A>G(rs689466), -765G>C(rs20417) in the promoter region are associated with risk of developing gastroesophageal cancer. The SNP 1759G>A(rs3218625) will induce 587Gly/Arg change had significant impact on COX-2 activity; the COX-2-Arg587 isoform showed 1.5 fold elevated enzymatic activity than the COX-2-Gly587 isoform in converting arachidonic acid into prostaglandins. The SNP 8473T>C (rs5275) in 3'UTR region are associated with risk of lung cancer.The aim of this case-control study including 260 head and neck squamous cell carcinoma cases and 1047 controls is to investigate the associations of the SNP -1195A>G, -765G>C in the cox-2 promoter regiong, the SNP 1759G>A in the coding region and the SNP 8473T>C in the 3'UTR regiong with head and neck squamous cell carcinoma risk in Chinese populations.We genotyped the four polymorphisms by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) and TaqMan-MGB method. In addition, genotyping was performed blindly and 10% of the samples were randomly selected for repeated assays. Finally, a total of 259 head and neck squamous cell carcinoma cases and 1035 controls were successfully genotyped.In the present case-control study, the genotype of -1195AG and -1195GG, were associated with no significantly increased risk of head and neck squamous cell carcinoma (adjusted OR=0.87, 95% CI=0.63-1.20; OR=1.02, 95% CI=0.69-1.50 ); compared with the wild-type genotype; the genotype of -765GC was not associated with the risk for developing head and neck squamous cell carcinoma (adjusted OR=1.01,95% CI=0.61-1.67); compared with the wild-type genotype, the genotype of 1759GA was not a risk factor for susceptibility to head and neck squamous cell carcinoma (adjusted OR=0.42, 95% CI=0.16-1.07 ); the genotype of 8473TC and 8473CC may be not the risk factors for developing head and neck squamous cell carcinoma (adjusted OR=0.90, 95% CI=0.66-1.22; OR=1.48, 95%CI= 0.68-3.25).These findings suggested that the four SNP may not associate with head and neck squamous cell carcinoma risk in Chinese population.Part II: Functional relationgship between SNP in the COX-2 promoter regiong and the changes of promote activityThe COX-2 promoter region has identified several key cis-acting regulatory elements to bind these transcription factors (such as PEA3, AP1. interleukin-6, nuclear factor KB, Sp1 and c-MYB), which may play a decisive role in the regulation of COX-2 transcription. The mutations within the consensus sequences of certain cis-acting elements significantly altered gene expression.In our research we constructed a reporter plasmid encompassing -1940 to +201 base pairs of human COX-2 promoter region. The constructs used in this study were restriction mapped and sequenced to confirm their authenticity. The PCR product was digested, site-directed mutated and ligated, respectively, into an appropriately digested pGL3-Basic vector (Promega) containing the firefly luciferase gene as a reporter. Then we transient transfected the Tca-8113, Hela and MG-63 cells with these re-construct plasmids .Accroding to the Luciferase Assays, we found -1195A-containing COX2 promoters were higher than those driven by the -1195G-containing counterparts(P<0.001). No significant effect of the -765G>C polymorphism on the promoter activity was observed under our experimental conditions. These results suggest that the -1195G>A SNP can influences COX-2 promoter activity, but the -765G/C containing haplotypes no associated with the promoter activity .