Association And Functional Studies Of HLA-G3’ UTR14-bp Insertion/Deletion Polymorphism With Hepatocellular Carcinoma Susceptibility In A Chinese Population

Objective: To investigate whether an Indel polymorphisms on the3′UTR of HLA-Gwas associated with hepatocellular carcinoma (HCC) susceptibility in a Chinese populationand to investigate the possible functional significance of this polymorphism usinggenotype-phenotype correlation as well as bioinformatics analysis.Methods:(1) Using bioinformatic tools to screen polymorphisms in HLA-G3′UTR,from which we chose a potential Indel polymorphism (rs66554220) as candidate.(2) Acase-control study consisting318HCC cases and599healthy controls was performedusing PCR-PAGE method. Logistic regression model was used for evaluating theassociation between polymorphisms and HCC susceptibility.(3) Immunohistochemical(ISH) analysis was used to investigate the genotype-phenotype correlation between thepolymorphism and expression of HLA-G in HCC tissue samples.(4) Bioinformatic methodwas used to predict the miRNA binding within rs66554220.Results:(1) Genotyping results showed that rs66554220had good genotypic andallelic frequency distributions. The allelic frequencies of insertion and deletion were0.234,0.766in case group,0.291,0.709in control group, respectively. There were no deviationsfrom Hardy–Weinberg equilibrium for both cases and controls.(2) Logistic regressionanalysis showed that after adjustment for sex, age, smoking status, drinking status andHBV infection and tumor stage, the14-bp Ins/Ins homozygote was associated with areduced risk of HCC compared with the14-bp Del/Del homozygote (OR=0.54,95%CI:0.30–0.98, P=0.031). At the allelic level, the14-bp Ins allele was also associated with areduced risk of HCC compared with the14-bp Del allele (OR=0.74,95%CI：0.59–0.93,P=0.009). Based on HBV stratification analysis, the differences between cases and controls were more obvious in the HBV-positive than the HBV negative population.(3) The ISHstudies revealed HLA-G expression in HCC tissues was [1.59(0.98–1.75)] in homozygous14-bp Ins/Ins genotype,[1.64(1.02–1.81)] in heterozygous, and [2.67(2.13–2.95)] inhomozygous14-bp Del/Del genotype, respectively. There was a significant differencebetween Ins/Ins and Del/Del genotypic groups concening HLA-G expression (P<0.01).(4)Bioinformatics analysis revealed that rs66554220site could bind25miRNAs, of which7miRNAs’ G difference were greater than4.00Kcal/mol. Hsa-miR-668andhsa-miR-557had the greater G (7.50Kcal/mol and5.34Kcal/mol, respectively).Conclusion:(1) Our genetic association studies have successfully identified the14-bpIndel polymorphism as a risk-conferring polymorphism for HCC in a Chinese population.Moreover, stratification analysis based on HBV infection showed that the association wasstronger in the HBV-positive individuals.(2) The immunohistochemical studies revealedthat the genotype of rs66554220is correlated the expression of HLA-G in HCC tissues.(3)(4) Bioinformatics analysis indicated that rs66554220may interrupt the binding of specificmiRNAs to HLA-G3′UTR, through which influence the expression of HLA-G, thusinvolved in the occurrence of HCC.