The Association Analysis Of Candidate SNPs With Lung Cancer And The Study Of Mechanism Of The SNP

Objective: Lung cancer incidence in China ranks first in the variety of tumors, but its pathogenesis is not completely clear. At present, several genes have been found to have a close relationship with lung cancer, such as: ras, myc, p53 and so on. In recent years, Genome-wide association study found that many new single-nucleotide polymorphisms(SNPs) is associated with lung cancer. Some these SNPs located in introns or intergenic region. The effect of these SNPs is unclear. Therefore, we selected six newly discovered SNPs which associated with lung cancer to genotype, screening SNP sites which associated with lung cancer in our study populations, and explore the SNPs effect.Methods: This study is divided into two parts. In the first part, the six candidate SNPs were genotyped using SNa Pshot technology in 391 cases of cases and 342 controls and then analyzed the association with lung cancer. In the second part, we overexpressed the SNAI1, and then luciferase assay was used to clarify the regulation function of rs2853677 regions to TERT expression. Next,Electrophoretic Mobility Shift Assay(EMSA) and Chromosome Immunoprecipitation(Ch IP) were used to detect the interaction between SNP site regions and protein. Finally, the conformation between the 1kb regions of rs2853677 and 500~1500bp downstream of transcription start site of TERT gene were analyzed using Chromosome Conformation Capture(3C).Results: In the first part, through genotype six SNPs of rs753955, rs2853677, rs2736100, rs4488809, rs2741354, rs12296850, the association analysis found that rs2853677, rs2736100 and rs12296850 sites related to occurrence of lung cancer(adenocarcinoma). allele C on rs2853677 and allele G on rs2736100 increase the risk of occurrence of lung cancer(adenocarcinoma), and allele G on rs12296850 decrease the risk of adenocarcinoma. In the second part, we selected rs2853677 site for further analysis. Lucifer assay indicates that the 1kb region around rs2853677 site exists an enhancer which regulates the expression of TERT. EMSA assay found that when the probe contains T allele on rs2853677, SNAI1 can bind to SNP regions, but SNAI1 can’t bind to the C allele. Ch IP assay also found that in H446 cells which the genotype of rs2853677 is T/T SNAI1 can bind to the SNP, and more, we found that when SNAI1 bind to the SNP, it can inhibit the activity of enhancer, thereby reducing the expression level of TERT gene. But in H209 cells which the genotype is C/C, SNAI1 can’t bind to the SNP. 3C technique confirmed that the rs2853677 site and TERT gene promoter region close to each other in space, and further verify the existence of enhancer in TERT gene.Conclusion: In this study, we found that in the Chinese population rs2853677 site associated with lung cancer(lung adenocarcinomas) occurrence, the C allele increases the risk of lung cancer(adenocarcinoma). The 1kb region around rs2853677 site exists an enhancer which regulates the expression of TERT. Meantime, rs2853677 site can service as a transcription factor binding sites of SNAI1, and the SNP may affect affinity of transcription factor SNAI1 binding to the region. Bind of SNAI1 to the site can effectively reduce the vitality of enhancer which exist in the second intron of TERT, thereby reducing the expression level of TERT gene.