To Explore The Biological Function Of Lung Cancer Risk Rsl663689 In The Non-coding Region Of Chromosome 10p14 Gene

In our country,with the aggravation of environmental pollution?smoking population growth and increased personal life pressure,the incidence and mortality rates of lung cancer continued to increase in recent years.Lung cancer is also the leading cause of cancer death in China.As complex diseases,environmental and genetic factors contribute to the process of lung cancer,but the mechanism of lung cancer pathogenesis is still unknown.In order to uncover the genetic factor in lung cancer development,we started from lung cancer associated single nucleotide polymorphisms(single Nucleotide Polymorphisms,SNP)and elaborated the functional molecular biological function of rs 1663689 by molecular biology and epigenetics technology.SNP(single nucleotide polymorphism)is the most common genetic variation that could be passed to the offspring.Rapid progress of Genome-wide Association Study(GWAS)has led to identification of many disease associated SNPs(tag-SNPs)in a variety of complex diseases.These diseases associated SNPs are located in the non-coding regions,so explore their biological functions in disease occurrence would be helpful to elaborate the mechanism of complex diseases' pathogenesis.The existing results showed that rsl663689 located in 10p14 noncoding region correlated with lung cancer susceptibility.However,rs 1663689 locates 908kb far away from its nearest gene GATA3,which increase the difficulty to study its biology function.Enhancer is the specific DNA sequence,usually a few hundred to two thousands base pairs and could enhance its target genes' transcription.Enhancer regions often enrich for H3K4me1 and H3K27ac histone modifications,regulate its target genes that may locate far away in linear distance by forming chromatin loops and recruiting transcription factors.SNPs within enhancers can affect its regulatory ability through changing the binding of variety transcription factors.Not a few Studies have shown disease associated SNPs within enhancers could change its regulatory ability or the frequency of the interactions between enhancers and its target genes.All the studies provided useful clues for the study of the lung cancer associated SNPs in non-coding region.In this project,we focused on the lung cancer susceptibility SNP(rs1663689)to explore its biological significance in the development of lung cancer by identifying SNP directly associated enhancer.We analyzed the genotype of rsl 663689 in four lung cancer cells(A549?H1299?95-C?95-D)and one bronchial epithelial cells(Beas-2B).The results showed the correlation of the genotype of rs 1663689 with lung cancer phenotype.After bioinformatics analyzing and several molecular biotechnology methods,we finally defined 10pl4-En as an enhancer especially in Beas-2B cells and the genotype of rsl663689 could affect the regulatory ability of 10p14-En.However,10p14-En did not possess enhancer ability in A549 and H1299 cells.To explore the biological function of 10p14-En and its target genes in lung cancer,firstly,we analyzed the mRNA levels of five genes(GATA3?TAF3?KIN17?ITIH2 and ITIH5)flanking within 2Mb up and downstream of rs1663689.The mRNA results in five cell lines showed tumor suppress gene ITIH5 was specific high in Beas-2B cells when compared with the other four lung cancer cell lines.The correlation is coincidence with the genotype of rs1663689 in Beas-2B and 4 lung cancer cell lines.Then,we captured the interaction between 10p14-En and the promoter of ITIH5 by Chromosome Conformation Capture technique.Furthermore,we knocked out 10p14-En in Beas-2b cells by CRISPR/Cas9 technique and the ITIH5 mRNA level was significantly decreased.ITIH5(Inter-a-trypsin inhibitor heavy chain 5)is a tumor suppressor gene,the main function is maintaining the stability of proteins of the extracellular matrix.Its low expression level in lung cancer cells may promote lung cancer migration.The expression level of ITIH5 in lung cancer tissue was significantly lower than normal control in TCGA database.We over-expressed ITIH5 in two lung cancer cell lines with low expression ITIH5 level significantly inhibited the invasion and migration of lung cancer cells.Our results demonstrate that the lung cancer risk SNP(rs1663689)mutant genotype(C)inhibits 10p14-En enhancer activity,decreased its target gene ITIH5 expression level,and promoted lung cancer cell migration.