The Difference Of RIG-I Expression Influence On IFN Therapeutic Response In CHB Patients And Its Mechanism

?Objective? 1. research the association between expression of rentinoic acid-inducible gene(RIG-I) and the response of IFN therapy in CHB patients. To investigate the values of common laboratory indexes and expression of RIG-I in predicting of efficacy of IFN in patients with CHB. 2. Through cell culture in vitro, research the relationship between RIG-I and IFN signal transduction pathway. Study the mechanism which RIG-I regulate JAK-STAT signal transduction pathway and stimulate the expression of antiviral protein, and provide fundamental basis for clinical individual therapy.?Method? 1. The quantitative of RIG-I can be used in predicting the IFN therapeutic response in CHB patients A Total of 65 CHB patients(44 of patients with HBe Ag positive and 21 of patients with HBe Ag negative) were recruited from The 1st Affiliated Hospital of Fujian Medical University between July 2013 and August 2015. All patients were treated with IFN weekly for 48 weeks and followed-up for another 24 weeks. We classified these patients into two groups(responders and no-responders) according to the efficacy of IFN. The concentration of HBV markers(HBs Ag and HBe Ag), HBV DNA level, ALT and AST were tested by Chemiluminescence microparticle immune assay(CMIA), real-time PCR and velocity method directly. Meanwhile, the expression of RIG-I was tested by quantitative real time PCR with specific primers. All statistical analysis were performed by normality test, t test and ?2 test,ROC analysis. The p values were calculated in SPSS 18.0. and the statistical significance level was accepted as p < 0.05. 2. RIG-I Enhances IFN-? Response by Promoting Antiviral Proteins Expression in Vitro. Interferon exerted its effect through JAK-STAT signal transduction pathway. In order to research the relationship between RIG-I and JAK-STAT signal, firstly, we performed the expression of antiviral proteins(ADAR1, Mx A, OAS, PKR) in response group and no-response group. Secondly, Hep G2 cell line was cultured with DMEM, and RIG-I was knocked out, then we performed expression of antiviral proteins above. Adding different concentration of interferons(0 u, 50 u, 100 u) in cell cultures, analyzed the change of antiviral proteins expression. Lastly, Combining the above two factors in Hep G2 cell lines, analysis of RIG-I genes in IFN regulatory mechanism of signal transduction pathways.?Results? 1. The quantitative of RIG-I can be used in predicting the IFN therapeutic response in CHB patients. At the end of treatment, 30 of 65 achieved responders and 35 of 65 achieved no-responders. For 42 HBe Ag positive patients, 12 patients achieved response and 30 patients achieved no-response. For 23 HBe Ag negative patients, the number of responder and no-responder are 18 and 5 respectively. For age and gender, there was no significant difference between responders and no-responders. We also found that there was no significant difference in HBV genotype, HBs Ag, HBV DNA, ALT and AST at baseline(p value was 0.523, 0.074, 0.073, 0.444 and 0.534 respectively). However, RIG-I expression was significantly higher in responders than in no-responders(p value was 0.014). In HBe Ag cohort, there was significantly difference between responders and no-responders merely(p value was 0.014). In HBe Ag negative cohort, there was no significantly difference in all indexes(the value of RIG-I expression was 0.053, close to 0.05). All currently dataindicated that the positive correlation between RIG-I expression and the response of IFN therapy in CHB patients. Patients with higher RIG-I expression could be better response to IFN therapy while patients with lower RIG-I expression could be worse response to IFN therapy. According to ROC curve, AUC of HBs Ag, HBV DNA and RIG-I expression was 0.711, 0.404, 0.286 respectively. 2. RIG-I Enhances IFN-? Response by Promoting Antiviral Proteins Expression in Vitro. There was significantly difference in antiviral proteins expression such as PKR, ADAR1, Mx A and OAS between response group and no-response group in patients with CHB(p value was 0.03, 0.012, 0.02 and.0.024 respectively). Meanwhile, the result of HBe Ag positive cohort and HBe Ag negative cohort are in agreement with the above analysis. Therefore, the expression of PKR, ADAR1 Mx A and OAS was regarded as efficacy of interferon. Then RIG-I gene was knocked out in Hep G2 cell lines, we found the expression of these antiviral proteins were decreased compared with Hep G2 cell lines which RIG-I gene was not knocked out. Then different concentrations of interferon were added into Hep G2 cell cultures solution, we found that the expression of antiviral proteins was increased compared with that culture solution did not add interferon. We also found that RIG-I expression increased after interferon was added into cluture solution. Lastly, we knock down RIG-I gene meanwhile adding IFN-?(Figure 4C~4F). Compared with non-IFN-stimulation, the expression of antiviral proteins is not significant difference in Hep G2 with IFN-stimulation after RIG-I gene was knocked down. However, only when RIG-I gene existed, the expression of antiviral proteins could decline after IFN stimulation. Taken together, we found that RIG-I is an important gene in IFN-induced antiviral activities and IFN signal transduction pathway.?Conclusion? 1. The quantitative of RIG-I can be used in predicting the IFN therapeutic response in CHB patients. There was statistical significance in RIG-I expression between responders andno-responders in CHB patients with IFN therapy. Patients with higher RIG-I expression could be better response to IFN therapy while patients with lower RIG-I expression could be worse response to IFN therapy. RIG-I was higher than any other laboratory indexes in specificity and sensibility. Therefore, RIG-I can be used in predicting the IFN therapeutic response in CHB patients. 2. RIG-I Enhances IFN-? Response by Promoting Antiviral Proteins Expression in Vitro. Interferon play the role of antiviral efficacy through expression of ADAR1, Mx A, PKR and OAS in JAK-STAT signal transduction pathway. RIG-I enhances interferon response by promoting antiviral proteins expression.