| Objective: To evaluate the effects of the short tandem repeat(STR) polymorphism(rs199618935) within 3’ untranslated regions(3’ UTR) of PTPN11 on hepatocellular carcinoma(HCC) susceptibility in Chinese populations and to investigate the possible functional significance of the polymorphism using genotype-phenotype correlation as well as bioinformatics analysis.Methods:(1) Using bioinformatic tools to screen polymorphisms in the 3’ UTR of PTPN11, from which we chose a potential short tandem repeat(STR) polymorphism(rs199618935) as candidate.(2) A case-control study consisting 705 HCC cases and 723 healthy controls was performed using PCR-PAGE method. Logistic regression model was used for investigating the association between the STR polymorphism and HCC susceptibility.(3) Real-time RT-PCR analysis were used to investigate the genotype-phenotype correlation between the STR polymorphism and expression of PTPN11 in HCC tissue samples, tumor adjacent tissue samples and HCC cell lines(Hep G2,Huh-7,Hep3B).(4) Constructed reporter plamids with the partial structures of PTPN11-3’ UTR containing allele 12,14 and 15 of rs199618935 was transfected into four hepatoma cell lines(Hep G2,Huh-7,Hep3 B and sk-Hep-1), Dual Luciferase reporter assay was used to further explore the genotype-phenotype correlation between the STR polymorphism and expression of PTPN11.(5) Effect of the STR polymorphism(rs199618935) on PTPN11 structure folding was predicted by bioinformatic method.Results:(1) Genotyping results showed that rs199618935 in 3’ UTR of PTPN11 had adequate genotypic and allelic frequency distributions. There were no deviations from Hardy–Weinberg equilibrium for both cases and controls(P>0.05).(2) Logistic regression analysis showed that after adjustment for sex, age, smoking status, drinking status and HBV infection and so on, compared with individuals who carry the genotype containing allele 11/12, those subjects of 13/14 genotypes had a significantly decreased risk of HCC(OR=0.72, 95% CI:0.59-.088, P =0.00101), with the risk decreased even further in those subjects of 15/16 genotypes(OR=0.51, 95% CI:0.37-0.69, P <0.0001). At the allelic level, the 13/14 or 15/16 alleles were associated with a reduced risk of HCC compared with the 11/12 alleles(OR=0.63, 95%CI:0.53-0.76, P <0.0001; OR=0.46, 95%CI:0.34-0.62, P <0.0001). Based on HBV stratification analysis, no obvious difference was observed between HBV positive and negative population.(3) Results of real-time PCR demonstrated that the expression level of PTPN11 in non-tumor tissues was 2.39 fold higher than that of HCC tumor tissues. The genotypes of rs199618935 polymorphism was significantly correlated with PTPN11 m RNA expression in vivo. Compared with 12-12 genotype, subjects carrying 14-14/14-15 genotype had 2.36 and 2.02 fold higher PTPN11 expression in HCC tumor tissues and adjacent non-tumor tissues, respectively. Compared with Hep G2 cell lines which carry 12-15 genotype containing a 12 allele, the PTPN11 m RNA expression levels of Huh-7 and Hep3 B carrying 14-14 genotype were significantly increased.(4) Dual Luciferase reporter assay results demonstrated that the reporter constructs containing allele 14 and allele 15 drove an increased reporter expression compared with the constructs containing allele 12 in all four hepatoma cell lines(Hep G2,Huh-7,Hep3 B and sk-Hep-1).(5) Bioinformatics analysis revealed that the STR polymorphism within PTPN11-3’ UTR contributes to hepatocarcinogenesis, possibly by regulating PTPN11 expression through affecting the folding structures of PTPN11.Conclusion:(1) rs199618935 polymorphism in 3’ UTR of PTPN11 was significantly correlated with HCC susceptibility in Chinese populations.(2) The positive genotype-phenotype correlation between the polymorphism and expression of PTPN11 was validated in both HCC tissues and tumor adjacent tissues.(3) rs199618935 polymorphism could regulate PTPN11 expression and furtherly participate in HCC carcinogenesis through affecting the folding structures of PTPN11. |
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