Study On The The Interactions Between Hepatitis B Virus Mutations And The Polymorphisms Of STAT3in The Occurrence Of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is an important cancer in China. Multiple factorssuch as host genetic susceptibility, environmental exposure, and chronic virus infectionparticipate in the stepwise process leading to HCC. Chronic HBV infection is thepredominant risk factor of HCC in China. HBV genotypes, HBV DNA level, HBeAgstatus, and HBV mutations have been significantly associated with the risk of HCC. HBVinfection is endemic in China, the subjects infected with HBV account for one-third of thatin the world, while HCC cases in this country account for more than50%of the world.Besides, the prevalence of HCC in Chinese populations in rest of the world is high,indicating that the genetic susceptibility of Chinese desendents plays a critical role in thedevelopment of HCC. Inflammation play an important role in HBV-inducedhepatocarcinogenesis and prognosis, signal transducer and activators of transcription3(STAT3) is the key molecule of JAK/STAT signaling pathway, through which thepolymorphisms of STAT3affect the expressing of the target gene, and has been associatedwith the occurrence of HCC. HBx protein activates STAT3and may affect the level of itthrough miRNA. HBx and STAT3may interact with each other in the development of HCC.Thus it is of great value to investigate the associations of HBV mutations andpolymorphisms of STAT3with HCC, and their interaction in the development of HCC.Objective: This study was aimed to investigate the associations of mutations in HBVEnhancer II/BCP/PC and preS region with Chronic hepatitis B (CHB), Liver cirrhosis(LC), and HCC, and the associations of STAT3polymorphisms (rs4796793, rs2293152,rs1053004) with susceptibility to HCC. To further elucidate the interactions between HCCassociated HBV mutations and polymorphisms of STAT3during the development ofHBV-related HCC, and reveal the viral and genetic risk factors for HCC, so as to provideclue in clarifying the mechanism of HBV-induced hepatocarcinogenesis and lay foundationon specific prophylaxis.Methods: A total of3023subjects, including1012healthy controls,990HBV-infectedsubjects without HCC (316asymptomatic HBsAg carriers [ASCs],316patients with CHB,and316patients with LC), and1021HCC cases were included in this study. MultiplexPCR was used to determine the genotypes of HBV from ASCs, CHB, LC, and HCC. TheEnhancer II/BCP/PC and preS gene of HBV were amplified by nested PCR with the products sequenced, and then the sequences were aligned and analyzed by using MEGA5.0and Bioedit7.0to detect HBV mutations. The polymorphisms of STAT3(rs4796793,rs2293152, rs1053004) were genotyped by Fluorescent probe-Real time quantitative PCR.Hardy-Weinberg equilibrium test was performed on the internet (http://ihg. gsf.de/ihg/snps. html). The SPSS16.0software was used to organize and analyze the data.Student's t test, chi-square test, and unconditional logistic model were used to elucidate theassociations of HBV mutations and polymorphisms of STAT3with HCC, and investigatethe interactions between HCC associated HBV mutations and STAT3polymorphismsduring the development of HCC.Results: The sequences of the Enhancer II/basal core promoter/precore (EnhII/BCP/PC)region of HBV were successfully obtained from252ASCs,172patients with CHB,224patients with LC, and512patients with HCC. The sequences of HBV preS region wereamplified from serum samples of130ASCs,164patients with CHB,194patients with LC,and460patients with HCC.1."Hotsopts" in the EnhII/BCP/PC and preS regions of HBVThe sites with mutation frequencies more than20%were termed as "hotsopts". The"hotspot" nt.1652, nt.1653, nt.1673, nt.1674, nt.1719, nt.1726, nt.1727, nt.1730, nt.1753,nt.1762, nt.1764, nt.1799, nt.1846, and nt.1896in the EnhII/BCP/PC region, and preSdeletion, nt.2857, nt.2875, nt.2931, nt.2946, nt.2964, nt.3026, nt.3051, nt.3054, nt.3060,nt.3063, nt.3066, nt.3069, nt.3116, nt.3120, nt.3186, nt.3191, nt.1, nt.7, nt.10, nt.31, nt.49,nt.52, nt.53, nt.76, nt.105, nt.109, nt.135, nt.147, and preS2start codon were included inthis study.2. Associations of mutations covering the EnhII/BCP/PC and the preS regions with CHB,LC, and HCC, respectively.(1) After the adjustment for age and sex, A1762m, G1764m, and12mutations in preS2region in genotype B were associated with increased risks of CHB, LC, and HCC, ascompared with ASCs, respectively. As for genotype C, G1719m, A1762m, G1764m,A1846m, G1896m, A1m, C7m, C10m, A31m, T49m, A52m, G105m, C109m, A135m,G147m, and preS2start codon mutation were associated with increased risks of CHB, LC,and HCC, as compared with ASCs following the adjustment for age and sex, respectively,while A1652m, C1673m, C3051m, C3063m were inversely associated with the risk ofCHB, LC, and HCC, as compared with ASCs, respectively. (2) After the adjustment for age and sex, C1673m, A1726m, A1727m, C1730m, G1764m,C1799m, and T2857m in genotype C significantly increased the risk of LC, as comparedwith the patients with CHB, while C3051m, C76m, and preS2start codon mutation ingenotype B decreased the risk of LC, as compared with the patients with CHB.(3) After the adjustment for age and sex, C1653m, T1674m, G1719m, T1753m, A1762m,G1764m, preS deletion, T2857m, C2875m, C2931m, A3120m, A3186m, G3191m, andT53m in genotype C significantly increased the risk of HCC, as compared with the patientswith CHB, and mutations at nt1726in both genotype B and C decreased the risk of HCC,as compared with the patients with CHB.(4) After the adjustment for age and sex, C1653m, T1674m, G1719m, T1753m, A1762m,C2875m, C3026m, A3120m, and C76m in genotype C were risk factors for HCC ascompared with the patients with LC, while the mutations at nt.1673, nt.1726, nt.1727, andnt.1730in both genotype B and C decreased the risk of HCC.(5) After the adjustment for age and sex, the mutations at nt.1652, nt.1719, nt.1726,nt.1727, nt.1764, nt.1896, and all mutations in preS2region except at preS2start codon,nt.53, and nt.76in both genotype B and C increased the risk of LC, as compared withASCs plus the patients with CHB.(6) After the adjustment for age and sex, C1653m, T1674m, G1719m, T1753m, A1762m,G1764m, A1846m, G1896m, preS deletion, T2857m, C2875m, C3026m, and A3186m ingenotype C significantly increased the risk of HCC, as compared withASCs, the patientswith CHB, and the patients with LC, respectively. The mutations in preS2except at preS2start codon, nt.7, and nt.53in both genotype B and genotype C were risk factors for HCC.(7) The mutations at nt.1653, nt.1719, and nt.1762increased the risks of HCC, whilemutations at nt.1652, nt.1673, nt.1726, and nt.1730decreased the risk of HCC after theadjustment for age and sex in HBeAg negative subjects, as compared with the patients withLC. No associations between the mutations and HCC were found in HBeAg positivesubjects. The mutation frequencies at nt.1653, nt.1674, nt.1846, and nt.1896weresignificantly higher in HBeAg negative than that in HBeAg positive HCC patients(P=0.000).3. Multivariate regression analysis for factors independently associated with the risk ofHCC and LC.(1) Independent factors contributing to HCC were evaluated in the forward stepwise multivariate regression analysis. T1674C/G (AOR=2.03,95%CI=1.34-3.07), A1762T/G1764A (AOR=2.74,95%CI=1.91-3.94), G1896A (AOR=2.05,95%CI=1.44-2.92) in theEnhII/BCP/PC region, and preS deletion (AOR=1.82,95%CI=1.14-2.91), preS2startcodon mutation (AOR=1.99,95%CI=1.20-3.30), C2875A(AOR=1.98,95%CI=1.17-3.36),C7A (AOR=4.96,95%CI=2.78-8.84), C76A (AOR=11.40,95%CI=4.80-27.06) in the preSregion as well as age, male, HBeAg negative, and genotype C were independentlyassociated with the risk of HCC.(2) The same model as for HCC were used to evaluate the independent risk factors forLC. A1762T/G1764A(AOR=2.37,95%CI=1.44-3.91), G1896A(OR=2.75,95%CI=1.64-4.60), preS deletion (AOR=4.96,95%CI=2.20-11.19), T2857C (AOR=6.98,95%CI=2.47-19.72), and T3026C (AOR=2.15,95%CI=1.12-4.11) as well as age wereindependently associated with the risk of LC.4. Associations between polymorphisms of STAT3and susceptibility to HCC were asfollowed.(1) STAT3rs2293152GG genotype was significantly associated with an increased riskof HCC (OR=1.30,95%CI=1.02-1.66), as compared with the healthy controls. rs1053004CT or C allele (CT+CC) significantly increased the risk of HCC (OR=1.26,95%CI=1.04-1.53; OR=1.22,95%CI=1.02-1.45) and HBV-related liver diseases without HCC(OR=1.33,95%CI=1.10-1.61; OR=1.26,95%CI=1.05-1.51), as compared with thehealthy controls, respectively.(2) After stratified by gender, rs4796793GG genotype in male subjects was significantlyassociated with increased risks of HCC and HBV-related liver diseases without HCC(OR=1.38,95%CI=1.01-1.88; OR=1.40,95%CI=1.00-1.95, respective ly), as comparedwith the healthy controls, respectively. rs1053004CT or C allele (CT+CC) in malesubjects significantly increased the risks of HCC (OR=1.27,95%CI=1.03-1.57; OR=1.27,95%CI=1.04-1.55) and HBV-related liver diseases without HCC (OR=1.33,95%CI=1.07-1.68; OR=1.29,95%CI=1.05-1.60), as compared with the healthy controls,respectively.In female subjects, rs2293152GG genotype was significantly associated with anincreased risk of HCC (OR=2.43,95%CI=1.38-4.29; OR=1.92,95%CI=1.12-3.31), ascompared with the healthy controls and HBV-related liver diseases without HCC,respectively. While rs1053004CC allele decreased the risk of HCC (OR=0.48,95%CI =0.23-0.97), as compared with the healthy controls. However, this result needs to bevalidated using larger sample size.(3) After stratified by HBV genotypes, rs2293152GG allelic carriage in HBV genotypeC was significantly associated with an increased risk of HCC (OR=1.67,95%CI=1.17-2.36), as compared with HBV-related liver diseases without HCC.5. Interactions between the polymorphisms of STAT3and HBV genotypes andmutations were as followed.(1) Interactions between the polymorphisms of rs2293152and HBV mutations in theEnhII/BCP/PC region: rs2293152CC genotype and "wild-type" of T1674C/G, A1762T/G1764A, or G1896A served as control, respectively, the combined effects of rs2293152GG allele with C/G1674, T1762/A1764, or A1896significantly increased the risks of HCCin genotype C (OR=3.27,95%CI=1.26-8.44; OR=7.61,95%CI=3.02-19.13; OR=4.63,95%CI=2.01-10.66), respectively. However, no significant interaction was found betweenthe polymorphisms of rs2293152and HBV mutations in the EnhII/BCP/PC region.(2) Interactions between the polymorphisms of rs1053004and HBV mutations in theEnhII/BCP/PC region: rs1053004TT allele and "wild-type" of T1674C/G, A1762T/G1764A, or G1896Aserved as control, respectively, the combined effects of rs1053004CTallele with C/G1674or T1762/A1764significantly increased the risks of HCC (OR=2.17,95%CI=1.17-4.03; OR=2.91,95%CI=1.67-5.06, respectively) in male subjects after theadjustment for age and HBV genotype. A significant interaction was found betweenpolymorphisms of rs1053004and T1674C/G (OR=2.53,95%CI=1.10-5.78). As for infemale subjects, the combined effects of rs1053004CT allele with C/G1674, T1762/A1764,or A1896also significantly increased the risks of HCC (OR=2.97,95%CI=1.06-8.33;OR=4.54,95%CI=1.76-11.72; OR=2.79,95%CI=1.21-6.43). However, no significantinteraction was found between polymorphisms of rs1053004and HBV mutations in theEnhII/BCP/PC region in female participants.(3) Interactions between polymorphisms of STAT3and HBV genotype: When HBVgenotype B and rs4796793CC, rs2293152CC, or rs1053004TT allele served as control,the combined effects of HBV genotype C with rs4796793GG, rs2293152GG, orrs1053004CT significantly increased the risks of HCC after adjustment for age, sex, andHBeAg status.(OR=1.77,95%CI=1.04-3.01; OR=3.22,95%CI=1.79-5.81; OR=1.79,95%CI=1.14-2.82, respectively). However, no significant interactions were found between HBV genotype and polymorphisms of rs4796793, rs2293152, or rs1053004.Conclusions:1. Age, male, HBV genotype C, negative HBeAg, T1674C/G, A1762T/G1764A,G1896A, preS deletion, preS2start codon mutation, C2875A, C7A, and C76A are riskfactors independently associated with the risks of HCC.2. Age, A1762T/G1764A, G1896A, preS deletion, T2857C, and T3026C are risk factorsindependently associated with the risks of LC.3. STAT3rs2293152GG allelic carriage is associated with increased risk of HCC. CT orC allele of rs1053004is associated with risks of chronic HBV infection. STAT3-1697GGand CT or C allele of rs105300is associated with risks of chronic HBV infection in men,and those who carried rs2293152GG have increased risks of chronic HBV infection andHCC after that in women, however, this result needs to be validated using larger samplesize.4. STAT3rs2293152GG increased the risk of HCC in HBV genotype C.5. T1674C/G significantly multiply interacted with polymorphism of rs1053004in thedevelopment of HCC in men.