Association Of Forkhead Box O Gene MicroRNA Related Snps With Hepatocellular Carcinoma And Function Validation Of Loci

ObjectivesFOXO (forkhead box O) is an important class of signal transductionmolecules. The activities of FOXO are regulated by a variety of mechanisms,and it plays an important role in the process of DNA repair, replication,recombination, cell cycle and cell apoptosis. It was found in recent years thatthe abnormal expression of FOXO gene is associated with the occurrence anddevelopment of various diseases, especialy tumours. This study was conductedto investigate the association between FOXO gene SNPs (single nucleotidepolymorphism) and the hereditary susceptibility to HCC (hepatocellularcarcinoma), and HCC related SNPs function assays.MethodsSNPs that may affect expression or function of the gene were screened by usingbioinformatics analysis firstly. The SNPs of the human FOXO gene familyFOXO1, FOXO3, FOXO4, and FOXO6gene were screened on the NIEHS(National Institute of Environmental Health Sciences) database SNPinfo WebServer (http://snpinfo.niehs.nih.gov/). SNPs that meet the requirements of the study containing rs17592236of FOXO1, rs4946936of FOXO3, and rs4503258of FOXO4were selected. The function prediction on the MIRanda database(http://www.microrna.org/microrna/home.do) for the above three loci indicatedthat they are all potential microRNA (miRNA) binding sites. We conducted ahospital-based case-control study including1049cases (HCC patients) and1052controls (non-tumor patients). The cases and controls were all born in Guangxi,and frequency matched in age, genders, and nationalities. The information of allthe participants was collected by field epidemiological investigation, and bloodsamples were collected for extracting genomic DNA and building of specimenrepository that will be used in genetic typing. High flux TaqMan MGB real-timefluorescent quantitative PCR technology was applied on genotyping forrs17592236of FOXO1, rs4946936of FOXO3, and rs4503258of FOXO4. Dataentry and consistency check were conducted with the Epi Data3.1software. TheSPSS20.0software was using for statistical analysis. If the distribution ofgenotypes in the controls is in line with the Hardy-Weinberg equilibrium (HWE)was tested through the Haploview4.2software. Measurement datas andclassification datas were analysed with t-test and chi-square test respectively.Unconditional logistic regression model was used to calculate odds ratio (OR)and95%confidence interval (CI), gene-environment interactions, andgene-gene interactions. Moreover, dual luciferase reporter gene function assayswere carried out to investigate the specific functions of SNPs which were foundassociated with HCC in the case-control study.Results(1) Basic information of the study subjectsThe distributions of age, gender, nation in the cases and the controls has nostatistical significance (P>0.05), equally comparable. However, the distributions of smoking, drinking, HBV infection and family history of HCC in the twogroups are statistically significant (all P<0.05).(2) Distribution of genotype frequencyTested by HaploView4.2, in the controls, the genotype distributions ofFOXO1rs17592236and FOXO3rs4946936are in the HWE (rs17592236:χ2=2.14, P=0.143; rs4946936: χ2=0.06, P=0.813). Because FOXO4rs4503258locates on the X chromosome, the test of HWE was not conducted.No statistical significance was found in the distributions of genotypes ofrs17592236, rs4946936and rs4503258between the cases and the controls(P>0.05). Multiariable Logistic regression analysis showed that rs17592236CT/TT genotype might reduce risk of HCC P=0.010，OR (95%CI)=0.699(0.526~0.927).(3) Stratification analysisThe results of stratification analysis on HBV infection and family history ofHCC indicated that SNPs of rs17592236and rs4503258are statisticallyassociated with HCC occurrence. No statistical association was found betweenHCC and SNP of rs4946936.(4) Gene-environment interactionsGene-environment interactions were found between rs17592236、rs4946936、rs4503258and environmental risk factors of smoking, drinking,HBV infection, and family history of HCC.(5) Gene-gene interactionGene-gene interaction existed between SNPs of rs17592236and rs4503258P=0.003, OR (95%CI)=0.755(0.628~0.908).(6) Function assaysThe result of dual luciferase reporter gene assays showed that the wild- type rs17592236had a stronger capacity of binding with miR–137than mutatedrs17592236. The relative luciferase activities are: FOXO1-wild,3.82±0.87;FOXO1-mut,22.83±3.97, P<0.001.ConclusionMutation of rs17592236might reduce the risk of HCC. The interactionsbetween rs17592236, rs4946936, rs4503258and environmental risk factorswere associated with HCC occurrence. Rs17592236is a binding site ofmiR–137, and mutation of the loci might reduce the affinity of miR–137binding to mRNA of FOXO1, ultimately resulted in reduced risk of HCC.