The Molecular Mechanism Study Of Paclitaxel-induced HBV Reactivation

Objective: 1.To verify the induction of HBV reactivation by paclitaxel at the cellular level in vitro;2.To explore the molecular mechanisms involved in HBV reactivation.Method: 1.HepG2-H4(Cells stably transfected with the HBV genome)and Hep AD38 cells(hepatocellular carcinoma cells with high levels of stable replication of hepatitis B virus)were selected as the study subjects,and HepG2 cells(hepatoma cells without hepatitis B virus replication)were used as blank controls.The experimental group was treated with paclitaxel at a concentration of 8 μM.PBS was used as a negative control and the change of HBV DNA copy amount before and after administration in cell model was detected by real-time PCR.2.The fluorescein reporter plasmid containing HBV core promoter Cp,X gene promoter Xp,surface antigen promoters Sp1 and Sp2 was constructed with p GL3-Basic as the vector;the experimental method using dual luciferase four promoter plasmids of PGL3-HBV(p GL3-Sp1,p GL3-Sp2,p GL3-Xp and p GL3-Cp)and co-transformed as an internal control p RL-CMV(renilla luciferase)and p GL3-control(positive control)Dye into HepG2 cells;Add paclitaxel 8μM or PBS to continue culture after 24 hours,the medium was changed to continue the culture and the cells were collected the next day.Then the activity of the HBV promoter was examined.3.HepG2-H4 and Hep AD38 cells were treated with paclitaxel,and PBS was used as a negative control.HepG2 cells were used as blank control.After 72 hours of treatment,real-time quantitative PCR was used to detect HBV replication.Relevant transcription factor expression levels.Result: 1.Paclitaxel can induce HBV reactivation in cell models with stable expression of HBV(HepG2-H4 and Hep AD38)in vitro.Real-time fluorescence quantitative PCR confirmed that the HBV DNA copy amount in the experimental group(paclitaxel)and in the supernatant of cell culture medium was significantly higher than that in the PBS treatment group(negative control group),showing a statistical difference(P<0.05).The results confirmed that paclitaxel could induce HBV reactivation.2.After treatment with paclitaxel,the activity of all four HBV promoters was changed,and the activity of promoter p GL3-Xp and p GL3-Cp was significantly up-regulated compared with that of PBS group(P<0.05).The activity of p GL3-Sp1 and p GL3-Sp2 promoters was not significantly affected compared with the control group.3.Among the 13 transcription factors involved in HBV replication,the expression levels of NF-κB(65),TR2,TR4,Coup-TF1 and CREB2 were decreased(P>0.05).The expression levels of the remaining transcription factors increased,and the transcription factor AP-1 increased most significantly(P<0.05),which was statistically different.Conclusion: 1.Paclitaxel can induce HBV reactivation at the cellular level in vitro.2.Paclitaxel up-regulates the activity of the HBV DNA promoters p GL3-Xp and p GL3-Cp.3.Paclitaxel up-regulates the expression level of HBV DNA transcription factor AP-1.