An Insertion/Deletion Polymorphism Within RERT-lncRNA Modulates Hepatocellular Carcinoma Risk

Objective: To investigate whether an Indel polymorphisms in RERT-lncRNA wasassociated with hepatocellular carcinoma (HCC) susceptibility in Chinese populationsand to investigate the role of this polymorphism in HCC carcinogenesis usinggenotype-phenotype correlation as well as molecular biological functional analysis.Methods:(1) A potential Indel polymorphism (rs10680577) was chosen fromRERT-lncRNA using bioinformatic tools.(2) A case-control study consisting1067HCCcases and1692healthy controls was performed using PCR-PAGE method. Logisticregression model was used for evaluating the association between polymorphisms andHCC susceptibility.(3) Realtime PCR and Western blotting analysis was used toinvestigate the genotype-phenotype correlation between the polymorphism andexpression of EGLN2in HCC tissue samples and five hepatoma cell lines samples.(4)Constructed plamids with putative promoter including insert or deletion allele ofrs10680577was transfected into five hepatoma cell lines, negative reports of DualLuciferase reporter assay detected the promoter regulating possibility.(5) Inconsideration of the location of locus, effect of the Indel polymorphism (rs10680577)on RERT-lncRNA second structure folding was predicted by bioinformatics analysis.(6)Constructed RERT-lncRNA vectors or empty vector were transfected into Hepatoma cells,expression of EGLN2and lncRNA was observed by Realtime PCR.Results:(1) Genotyping results showed that rs10680577had adequate genotypicand allelic frequency distributions. The allelic frequencies of insertion and deletion were0.759,0.241in case group,0.819,0.181in control group, respectively. There were nodeviations from Hardy–Weinberg equilibrium for both cases and controls. Logisticregression analysis showed that after adjustment for gender, age(median), smokingstatus, drinking status and HBV infection, the4-bp Del/Del homozygote was associated with a increased risk of HCC compared with the4-bp Ins/Ins homozygote (OR=2.32，95%C.I.:1.54-3.49，P<0.0001). At the allelic level, the4-bp Del allele was alsoassociated with a reduced risk of HCC compared with the4-bp Ins allele (OR=1.44，95%C.I.：1.26-1.64，P<0.001). Based on smoking status stratification analysis, thepositive association was more pronounced in current smokers (Heterogeneity P=0.0002).(2) The genotype–phenotype correlation between rs10680577and EGLN2expressionshowed that EGLN2mRNA level in samples with ins/del and del/del genotype wassignificantly higher than that in the ins/ins genotype.In terms of RAB4B, no significantdifference was observed between2genotypic groups.Further genotype–phenotypecorrelations in4common hepatoma cell lines (HepG2, Hep3B, sk-Hep-1, andSMMC-7721) and L02hepatic cell line betrayed that the EGLN2mRNA expressionlevel in Hep3B with del/del genotype was significantly higher than the average level ofother4cell lines carrying ins/ins genotype. For both liver tissues and cell lines, westernblot results showed that EGLN2protein level of del/del genotype carriers was higherthan that with ins/del or ins/ins genotypes.(3) Dual Luciferase reporter assay resultsdemonstrated that there was no significant difference between the2constructscontaining either insertion ordeletion allele of rs10680577in any of the5cell linestested.(4) Bioinformatics analysis revealed that RERT-lncRNA secondary structurefolding difference could be caused by rs10680577polymorphism, the requiredminimum free energy changed. The expression levels of EGLN2and RERT-lncRNAwere significantly correlated in HCC tissue samples (R~2=0.540, P <0.001) and inhepatoma cell lines (R~2=0.823, P=0.034).(5) The constructedRERT-lncRNA-pcDNA3.1(+) plasmid or empty vector pcDNA3.1(+) were transfectedinto HepG2, SMMC-7721, and sk-Hep-1cells, the in vitro RERT-lncRNAgain-of-function analysis results showed that compared with cells transfected with emptyvectors, cells transfected with constructed RERT-lncRNA vectors displayed4.73-to9.82-fold expression of RERT-lncRNA, and1.61-to1.75-fold expression of EGLN2.Conclusion:(1) rs10680577polymorphism in RERT-lncRNA was significantlycorrelated with HCC susceptibility in Chinese populations, the positive association wasmore pronounced in current smokers.(2) The positive genotype-phenotype correlation between the polymorphism and expression of EGLN2was validated in HCC tissuesamples and five hepatoma cell lines samples.(3) rs10680577polymorphism couldregulated EGLN2expression and furtherly participate in HCC carcinogenesis throughaffecting RERT-lncRNA expression.