Association Between The Indel Polymorphism In The Promoter Region Of LncRNA GAS5 And Hepatocellular Carcinoma Susceptibility

Objective: To evaluate the effects of the Insertion/Deletion(Indel) polymorphism(rs145204276) within the promoter region of Lnc RNA GAS5 on hepatocellular carcinoma(HCC) susceptibility in a Chinese population and the possible functional significance of the polymorphism in carcinogenesis of HCC using genotype-phenotype correlation as well as bioinformatics analysis and epigenetic assays.Methods:(1) Using bioinformatic tools to screen polymorphisms in the promoter region of Lnc RNA GAS5, from which we chose a potential functional Indel polymorphism(rs145204276) as a candidate.(2) A case-control study including 1034 HCC cases and 1054 healthy controls was performed using PCR-PAGE(polyacrylamide gel electrophoresis) method. Logistic regression was used to analyze the association between rs145204276 and HCC susceptibility.(3) Real-time PCR analysis was used to investigate the genotype-phenotype correlation between different genotypes of the Indel polymorphism and the expression level of Lnc RNA GAS5 in the corresponding HCC tissue samples.(4) Constructed reporter plamid vetors with the fragment about 300 bp including the Ins/Del allele of rs145204276(p GL3-GAS5-WT/MT) in the upstream of GAS5 transcript start point were transfected into three hepatoma cell lines(Huh-7, BEL-7404 and SK-HEP-1), Dual Luciferase reporter assay was used to further explore the influence of rs145204276 on Lnc RNA GAS5 transcription activity.(5) Flow cytometry and Western Blot were used to examine the effect of overexpression/knockdown of Lnc RNA GAS5 on apoptosis in hepatoma cell lines.(6) The methylation status of the Cp G inlands of GAS5 promoter region in different genotypes of rs145204276 was assayed by quantitative bisulphite pyrosequencing.Results:(1) Genotyping results showed that the Indel polymorphism(rs145204276) in the promoter region of Lnc RNA GAS5 had adequate genotypic and allelic frequency distributions. The genotypic frequencies of rs145204276 were 0.478(Ins/Ins), 0.444(Ins/Del), 0.078(Del/Del) in control group, respectively; The allelic frequencies of insertion and deletion were 0.70, 0.30 in control group, respectively. There were no deviations from Hardy-Weinberg equilibrium(P>0.05). Logistic regression analysis showed that after adjustment for sex, age, smoking status, drinking status and HBV infection, compared with individuals carrying the Ins/Ins genotype, the individuals with the Ins/Del and Del/Del genotype were associated with an increased risk of HCC(OR=1.23, 95%CI: 1.02–1.48, P<0.05; OR=2.10, 95%CI: 1.54–2.87, P<0.0001).(2) The results of Real-time PCR demonstrated that the expression level of Lnc RNA GAS5 with Del/Del genotype was 2.55 fold higher than that with Ins/Del and Ins/Ins genotype in HCC tumor tissues.(3) In Dual Luciferase reporter assay, the results demonstrated that the luciferase activity of the cells transfected with p GL3-GAS5-WT or p GL3-GAS5-MT was higher than that of cells transfected with control constructs. Furthermore, the luciferase activity of the cells transfected with p GL3-GAS5-MT was higher than that of the cells transfected with p GL3-GAS5-WT in all of the three hepatoma cell lines(Huh-7, BEL-7404 and SK-HEP-1).(4) The results from flow cytometry analysis revealed that the rate of apoptosis in GAS5-overexpressed groups was significantly lower than that in control group(36.52±0.97% vs. 47.95±1.26%, P <0.001); Western Blot results showed that the expression level of caspase 3(an apoptosis-associated key player) was lower in GAS5- transfected group than that in control group. In contrast, the expression level of Bcl-2(a survival-associated crucial mediator) was higher in GAS5-transfected group than that in control group, while be knocked down of GAS5, the antiapoptosis effect of GAS5 could be at least partially reverted.(5) In pyrosequencing, the seventh site next to the indel polymorphism rs145204276, the mean methylation index of del/del genotype was markedly higher than that of either ins/del genotype or ins/del genotype(the mean methylation index was 41.3% for del/del genotype, 24.6% for ins/del genotype, 21.1% for ins/ins genotype; P<0.01).Conclusion:(1) rs145204276 polymorphism in the promoter region of Lnc RNA GAS5 was significantly correlated with HCC susceptibility in the Chinese population.(2) The positive genotype-phenotype correlation between different genotypes of the polymorphism and expression of Lnc RNA GAS5 in the corresponding HCC tissues was validated.(3) Lnc RNA GAS5 may play an antiapoptosis role in HCC cell lines.(4) rs145204276 polymorphism may play a functional role in carcinogenesis of HCC through regulating methylation status of the Cp G islands in the promoter region of Lnc RNA GAS5 and subsequently affecting the expression of GAS5.