Effects Of Interferon Alpha On Huh-7 Cells Transfected With HBV X Gene And The Predictors Of Anti HBV Response To Interferon Alpha

Background and Objectives:Interferon alpha(IFN-α)is currently used as a first-line antiviral drug to treat chronic hepatitis B.Interferon alpha is an antiviral cytokine that has a broad spectrum of action,exhibits high activity and indirect and species specificity.IFN-α exerts its antiviral activity via activation of the Janus kinase/signal transducer and activator of transcription(JAK-STAT)signaling pathway.The binding of IFN-α to its receptor IFNAR1 activates Janus kinase-JAK1 and non-receptor tyrosine-protein kinase TYK2,which leads to the phosphorylation of signal transducer and activator of transcription(STAT)1 and STAT2.STAT1 and STAT2 then form a heterodimer and bind to interferon regulatory factor 9(IRF-9)to form ISGF3.ISGF3 translocates from the cytoplasm to the nucleus and binds to the IFN stimulated regulatory element to promote the transcription of antiviral genes,such as PKR and RNaseL.In addition,interferon alpha also has biological functions such as anti-tumor,immune regulation and so on.The occurrence of primary liver cancer in 50-55% is caused by persistent hepatitis B virus(HBV)infection.Hepatitis B X protein(HBx),encoded by HBV DNA,serves an important role during the development of chronic hepatitis B,liver cirrhosis and liver cancer.Therefore,the current study established a novel HBV-related liver cancer model by transfecting the hepatoma cell line Huh-7 with HBx-expressing lentivirus,and subsequently investigated the effect of IFN-α on the cellular behavior and the expression of JAK-STAT signal pathway related molecules of the cancer cells.In addition,our study observed the predictive value of JAK-STAT pathway related molecules on the response of IFN-α in the treatment of chronic hepatitis B.Methods:1.We produced HBx-expressing lentivirus and then transfected it into Huh-7 cells.Subsequently we used an MTT assay to detect the cell viability,cell scratch test to detect the cell migration ability and cell invasion test to detect the cell invasiveness.After IFN-α was added to Huh-7 and Huh-7-HBx,the effects of IFN-α on the biological behavior of the above cells were observed.2.We transfected HBx-expressing lentivirus into Huh-7 cells and then added IFN-α.Subsequently we used RT-qPCR and Western-blot to detect the changes of expression level of related molecules on JAK-STAT signaling pathway before and after IFN-α intervention in above cells.3.Patients with chronic hepatitis B who met the indications of interferon alpha treatment were collected.The patients in the group were given polyethylene glycol IFN alpha-2b,1 g/kg/ weeks,1 times a week,subcutaneous injection,or regular IFN alpha,5 million U,once every other day,subcutaneous injection.The basic course of treatment was 48 weeks,and the treatment of non responders was stopped at 24 weeks.We collected PBMCs at baseline(before treatment)and treatment for 4,8,12,and 24 weeks of these patients.The expression level of IFNAR1,IFNAR2,ISGF3,PKR and RNase-L in PBMCs were detected by RT-qPCR and Western blot respectively.Finally,the differences of the above molecular expression levels in PBMCs between the effective and ineffective groups were statistically analyzed.Results:1.The effect of IFN-α treatment alone on Huh-7 cell viability compared with the control group was not significant.Similarly,the difference in cell viability in the Huh-7-HBx and Huh-7-HBx+IFN-α treatment groups compared with the control was not significant.Cell migration was decreased(P<0.05)in the IFN-α treatment alone group and increased(P<0.05)in the Huh-7-HBx group compared with the control.There was a decrease(P<0.05)in cell migration in the Huh-7-HBx+IFN-α treatment group compared with the control,but not to the same extent as the decrease observed in the IFN-α treatment alone group.Huh-7 cell invasion was decreased(P<0.05)in the IFN-α treatment alone group and increased(P<0.05)in the Huh-7-HBx group compared with the control.There was a decrease(P<0.05)in cell invasion in the Huh-7-HBx+IFN-α treatment group compared with the control,but not to the same extent as the decrease observed in the IFN-α treatment alone group.2.RT-qPCR demonstrated that the expression of IFNAR1,IFNAR2,PKR,RNaseL and ISGF3 mRNA was significantly increased(P<0.05)in the IFN-α treatment only group compared to the control.mRNA levels of these antiviral genes were upregulated(P<0.05)in the Huh-7-HBx group compared with the control group however,not to the extent of the increase demonstrated in the IFN-α treatment only group.The most significant increase in expression of antiviral gene mRNA,compared with the control,was in the Huh-7-HBx+IFN-α treatment group.The protein expression of these antiviral genes was consistent with this.3.A total of 41 patients were enrolled,including 30 males and 11 females,18 patients were treated with Peg IFN alpha-2b,1.0μg/ kg,1 times a week;23 patients underwent conventional IFN alpha-2b,5 million units,3 times a week,all patients completed 24 weeks of treatment.At the end of the 24 week treatment,according to the above criteria,23 of the 36 patients were treated as effective group(group A),and 13 were treated as ineffective group(group B).At baseline,4 weeks,8 weeks,12 weeks and 24 weeks,mRNA expression level of IFNAR1,IFNAR2,ISGF3,PKR and RNASE-L were statistically significant in two groups,and all were A group >B group.At 4 weeks of treatment,the mRNA level of IFNR2 and PKR in the effective group was significantly higher than that in the ineffective group.At the 8 week treatment,the level of IFNR1 and ISGF3 in the effective group was significantly higher than that in the ineffective group,and the above protein levels detected by Western-blot showed the same results.Conclusions:1.HBx could increase the migration and invasion ability of Huh-7 cells in vitro,but had no significant effect on the viability,and IFN-α may inhibit cell migration and reduce cell invasion in HBV-related liver cancer.2.IFN-α can increase the expression of JAK-STAT pathway molecules IFNAR1,IFNAR2,ISGF3,PKR,RNase L in hepatocellular carcinoma cell Huh-7,and IFN-α can further increase the expression levels of the above molecules in Huh-7 cells transfected with HBx-expressing lentivirus.3.The differential expression of JAK-STAT pathway molecules IFNAR1,IFNAR2,IRF9,ISGF3 and RNASE-L genes and protein between groups and time points may be an important mechanism that affects the antiviral effect of IFN-α.From the point of view of the early predictive value of IFN-α in the response to chronic hepatitis B,IFNR2 and PKR are expected to be an early prediction of IFN-α response.