Exploring TRIM Gene Polymorphism Affecting The IFN-? Efficacy In CHB Patients And Its Mechanism

?Objective?1.In recent years,some members of the Tripartite motif?TRIM?family have received great attention due to their antiviral properties,single nucleotide polymorphism?SNP?is important for individual disease susceptibility and drug reaction differences based on Genetics.we aim to exploring the key loci of TRIM gene which associate with the IFN-?efficacy of CHB patients,and provide the basis for individualized diagnosis and treatment.2.To explore the role of TRIM family proteins in IFN-?against HBV replication,and provide evidence for the mechanism of IFN-?anti-HBV replication.?Method?1.The whole blood and serum samples of 124 HBeAg-positive CHB patients who received IFN-?treatment?including IFN-?or PegIFN?monotherapy,combination or sequential therapy with IFN-?and NAs?were collected of in the Hepatology Center of the First Affiliated Hospital of Fujian Medical University from September 2015 to September2018.According to the efficacy at the end of the 48-week course,the Complete Response?CR?group was defined as HBV DNA<500 IU/ml,HBeAg negative or serological conversion,and/or HBsAg clearance.Suboptima Response?SR?was defined as HBeAg positive with HBV DNA decreased by?1 log₁₀0 IU/ml but still more than 500 IU/ml.whole blood DNA was extracted by spin column method,SNP typing via Infinium?Asian Screening Array high-throughput chip.HBV serum markers were detected by chemiluminescence microparticle immunoassay?CMIA?,HBV DNA was detected by real-time fluorescent quantitative PCR?RT-qPCR?,and ALT was detected by rate method.Statistical analysis used normality test,t test,?² test and so on.2.The difference in TRIM mRNA expression in whole blood of the CR group and the SR group was compared by RT-qPCR.At the same time,the TRIM mRNA expressions of different genotypes of TRIM SNPs analyzed by the gene chip was verified,and the correlation between TRIM SNPs and clinical indicators related to IFN efficacy was analyzed.3.The expression level of TRIM mRNA in HepG2.2.15 was detected by 10³ IU/ml IFN-?stimulation at different time points.The pHBV-1.2 expression vector and the pREP10vector were transfected into HepG2 cells stimuated with 10³ IU/ml IFN-?via Lipo8000transfection kit,and the expression level of TRIM22 mRNA and protein was detected by RT-qPCR and Western blot respectively.?Results?1.Relationship between TRIM gene polymorphism and IFN-?efficacy in HBeAg-positive CHB patientsA total of 483500 loci were detected in 124 samples using the Asian Screening Array and Massarray methods.After chi-square test and multiplexed test,23672 SNPs with P<0.05,4381 SNPs with P<0.01?1265 genes?and 51 SNPs with P<10^-5?20 genes?were obtained.The TRIM family gene P<0.05 involves 11 genes and 48 SNPs.By reviewing the literature and selection criteria for the minimum allele frequency?MAF??5%,we identified six SNP loci that are most likely to be associated with IFN-?efficacy in CHB patients.we found that the distribution of TRIM22 rs10838543 allele A,AA genotype and TRIM31 rs2523992 allele A,AA genotype are more prevalent in the SR group than the CR group?P<0.05?.TRIM15 rs2074477 allele G,GG genotype,TRIM26 rs12175655allele G,GG genotype and TRIM39 rs2240058 allele A,AA genotype were statistically significant,being more prevalent in the CR group than the SR group?P<0.05?.TRIM38rs62394537 allele G showed significant differences between CR group and SR group,but its genotype has no predictive value for the efficacy of IFN-?.TRIM15 rs2074477 GG genotype,TRIM39 rs2240058 AA genotype was positively correlated with HBeAg clearance?P<0.05?,TRIM22 rs10838543 AA genotype and TRIM31 rs2523992 AA genotype were negative correlated with HBeAg clearance?P<0.05?.There was no statistically significant difference in HBeAg serological conversion between the genotype of TRIM26 rs12175655 GG and the TRIM38rs62394537 GG were not associated with serological conversion of Hepatitis B virus e antigen.2.Relationship between TRIM gene polymorphism and TRIM mRNA expressionReal-time quantitative PCR results show that TRIM22 mRNA in the peripheral blood of patients with TRIM22 rs10838543 AA genotype is significantly lower than that of AG+GG genotype,while TRIM15/26/31/38/39 SNPs genotypes did not affect the corresponding TRIM mRNA expression.In addition,compared with the SR group,only TRIM22 mRNA expression was significantly increased in the CR group.3.Indicators predicting the efficacy of IFN-?In view of the association between TRIM SNPs and IFN-?efficacy,we retrospectively analyzed the current clinical routine antiviral indicators,it was found that the concentration of HBsAg and HBeAg at week 12 was statistically different between SR group and CR group.The ROC curve was used to analyze the predictive value of HBeAg,HBsAg and TRIM SNP genotype on IFN-?efficacy,and the areas under the HBeAg level and HBsAg concentration curve were 0.761?P=0.001?and 0.646?P=0.019?,respectively.At week 12,HBsAg<3.20 log₁₀0 IU/ml,HBeAg<2.57 log₁₀0 S/CO had a better chance of achieving a complete response at the end of the treatment.In addition,the area under the ROC curve of TRIM15 rs2074477?GG?,TRIM22 rs10838543?AA?,TRIM26 rs12175655?GG?,TRIM31 rs2523992?AA?,TRIM39 rs224005?AA?and the combined detection were 0.627?P=0.03?,0.680,respectively.?P=0.002?,0.589?P=0.13?,0.606?P=0.07?,0.621?P=0.038?,and 0.648?P=0.011?,The area under the curve of TRIM22 is up to 0.680.Using logistics regression analysis to predict the combined value of TRIM22,12-week HBsAg concentration and 12-week HBeAg concentration respectively,the AUC of the combination of TRIM22 and 12-week HBeAg concentration was 0.807[(P<0.0001,95%CI?0.718-0.878?],which was significantly better than the combined value of other indicators and a single indicator.4.The significance of TRIM in IFN-?against HBVThe expression of TRIM22 was lower in HepG2.2.15 and HepG2 cells,while TRIM22 mRNA was significantly increased by the stimulation of 10³ IU/ml IFN-?in in vitro,However,TRIM15,TRIM26,TRIM31,TRIM38 and TRIM39 showed no significant change.The results of Real-time quantitative PCR and Western-Blot shows that the level of TRIM22 was downregulated in 10³ IU/ml IFN-treated HepG2 cells transfected with HBV 1.2 expressnion vectors compared to 10³ IU/ml IFN-treated HepG2cells transfected with pREP10 vectors.This result suggests that HBV can inhibit the expression of TRIM22 in HepG2 cells.?Conclusion?1.TRIM gene polymorphism is associated with IFN-?efficacy in HBeAg-positive CHB patients.TRIM22 rs10838543?GG+AG?may upregulated TIRM22 mRNA level,which may affect therapeutic responses to chronic HBV infection.2.TRIM22 may be an important molecule in the anti-HBV effect of IFN-?,and the expression of TRIM22 may be affected by HBV replication activity.