The Functional Characterization Of Genetic Variants And Susceptibility Genes In Pancreatic Cancer Identified By Genome-wide Association Study

Pancreatic cancer is an uncommon malignancy of the digestive system.Recent years,its incidence rate has increased significantly.Genome-wide association study(GWAS)is an effective tool to study complex diseases.In our previous GWAS on pancreatic cancer,we identified 5 new susceptibility locus in Han Chinese from 2972 pancreatic cancer patients and normal controls(981 pancreatic cancer patients,1991 normal controls),and rs372883(3’UTR in gene BACH1)is the most significantly locus associated with pancreatic cancer susceptibility(T>C,odds ratio(OR)= 0.69,95%confidence interval(CI),0.62-0.70;P = 2.10 × 10-10),suggesting that BACH1 may play an important role in pancreatic cancer susceptibility.However,this is only the association analysis results,what we really want to know is the biological mechanism of susceptibility locus or genes in the development of pancreatic cancer.Therefore,in the present study we report the biological function of rs372883 and BACH1 in pancreatic cancer susceptibility by using sets of biochemical assays.We constructed the reporter gene vector containing rs372883 different genotypes.Combined with bioinformatics analysis,we found miRNA-1257 could bind to the rs372883T allele,while the C allele disrupts the binding of miRNA to mRNA.The binding of miRNA-1257 to the rs372883T allele significantly reduced the expression of the reporter gene,and this phenomenon could be reversed by inhibitors of miRNA-1257.We then examined the expression of BACH1 in surgically removed normal pancreas tissues adjacent to the tumors and found that the expression of BACH1 was significantly lower in patients with rs372883T allele than in patients with C allele.We further explored the biological effects of BACH1 gene on the development of pancreatic cancer.We investigated the expression of BACH 1 in pancreatic cancer and its adjacent tissues at mRNA and protein levels.The results showed that BACH1 is down-regulated in cancer tissue,suggesting that BACH1 may have anti-tumor potency.By constructing stably over-or down-expressed BACH1 in pancreatic cancer cell lines,we found that overexpression of BACH 1 significantly inhibited the proliferation,colony formation,and tubing formation of HUVEC in vitro,whereas down-regulation of BACH1 resulted in opposite results.In vivo experiments showed the similar results with in vitro studies.It has been previously reported that BACH1 is a transcriptional repressor and heme oxygenase 1(HMOX1)is its target gene.We found that BACH1 can bind to two enhancer regions upstream of HMOX1 by chromatin immunoprecipitation.The results of cell and tissue tests showed that BACH1 was negatively correlated with HMOX1 expression,HMOX1 could reverse the phenotype of BACH1.Further signal pathway analysis,we found that BACH1 regulate AKT and ERK-mediated cancer promoting pathway via HMOX1.Gemcitabine is the first line chemotherapy given to patients with advanced or unresectable pancreatic cancer,and previous studies have linked HMOX1 overexpression to resistance against gemcitabine in pancreatic cancer cells.Our results indicate that BACH1 is negatively correlated with HMOX1 and rs372883 polymorphism can influence BACH1 expression.Therefore,we hypothesized that rs372883 polymorphism would influence the sensitivity of gemcitabine and further impact the survival time of pancreatic cancer patients.We collected 102 pancreatic cancer patients treated with gemcitabine.The results suggest that patients with rs372883CC genotype are more sensitive to gemcitabine therapy than patients with TT genotype,and the survival time is significantly longer.In vitro cytotoxicity test also showed that pancreatic cancer cells with rs372883CC genotype treated with gemcitabine was less sensitive than TT genotype in the presence of miRNA-1257.These results suggest that rs372883 is not only associated with pancreatic cancer susceptibility,but also associated with the sensitivity of gemcitabine treatment.In conclusion,these findings explain the mechanisms of BACH 1 in maintaining cell homeostasis and functional germline variation may confer susceptibility and drug resistance to pancreatic cancer.Our findings are of importance in better understanding etiology of pancreatic cancer and may have potential utility in evaluating prognosis and determining gemcitabine response in clinical therapy.