Relationship Between CccDNA Transcription Activity And HBV PgRNA? DNA?and HBsAg Dynamics After NAs Treatment In Vitro Study

Objective To explore the dynamic relationship between cccDNA transcription activity and HBV pgRNA,DNA and HBsAg after NAs treatment,and to explore the feasibility of the pgRNA as a new marker of cccDNA transcriptional activity.Methods To establish an in vitro cell model of TDF antiviral therapy in HepG2.2.15 cells.The cells and supernatants were collected at baseline,medications 12 d,24d and 36 d.Total DNA was extracted from the cells,and HBV cccDNA levels were quantified by PSAD digestion,rolling circle amplification,and real-time fluorescence PCR.Alu-PCR was used to detect intracellular HBV S integration genes;Supernatant HBV pgRNA and HBV DNA levels were detected by real-time fluorescence PCR;Supernatant HBsAg levels was determined by chemiluminescence reagent.To evaluate the functional stability of HepG2.2.15 cell model without antiviral treatment;Depicting the time-content curve of each marker after TDF antiviral,to analyze the trends of HBV pgRNA,HBsAg,HBV DNA,and cccDNA levels as the treatment time prolongs.The bivariate correlation analysis was applied to analyze the correlation between changes in supernatants HBV pgRNA,HBsAg,HBV DNA and changes in intracellular cccDNA at each detection points.Results(1)The levels of supernatant HBV DNA and intracellular cccDNA in HepG2.2.15 cells without TDF antiviral therapy was the highest at baseline,The supernatant HBsAg levels reached the peak of secretion at 12 th day,and the levels of intracellular cccDNA and supernatant DNA,HBsAg is stable within 36 days(F=0.604,0.823,and 1.284,respectively,all P>0.05).The supernatant HBV pgRNA levels reached the peak of secretion at the 12 th day of culture and increased within 36 days(F=8.140,P<0.05).(2)The total levels of intracellular HBV cccDNA and supernatant HBV DNA,pgRNA,HBsAg,in HepG2.2.15 cells after TDF antiviral treatment showed a decreasing trend.HBV DNA first decreased below the detection limit,followed by HBsAg,while the levels of supernatant pgRNA and intracellular HBV cccDNA decreased slowly,both of them were not below the detection limit after antiviral treatment for 36 days.(3)HBV S integration gene was not detected in HepG2.2.15 cell at baseline and after antiviral treatment for 24 days.(4)After the TDF antiviral,the levels of intracellular cccDNA and the supernatant pgRNA showed the same downward trend,both of them were not below the detection limit within 36 days;From the baseline to treatment 12 d,12d to 24 d,24d to 36 d,there was a complete positive correlation between the two changes(r=1.000,p=0.000).(5)After TDF antiviral,the levels of intracellular cccDNA and supernatant HBsAg,HBV DNA levels showed a decreasing trend.HBsAg and HBV DNA Sustained decline and below the detection limit within 36 days,while cccDNA could still detect low positive value;There was no correlation between the changes of cccDNA and HBsAg from the baseline to treatment 12 d,12d to 24 d,24d to 36 d,(r=0.213,-0.016,and 0.593,respectively,all p>0.05).Similarly,there was no correlation between the changes of cccDNA and DNA.(r=-0.638,0.550,and 0.644,respectively,all p>0.05).Conclusions(1)The TDF anti-virus HepG2.2.15 cell model can reflect the relationship between cccDNA transcription activity and expression product after NAs therapy.(2)pgRNA can reflect the intracellular cccDNA levels and transcription activity better than HBsAg and HBV DNA,which is beneficial for monitoring the therapeutic effect of NAs therapy.(3)pgRNA detection is simple and non-invasive,and has broad clinical application prospects as an indicator of NAs therapy for monitoring the transcriptional activity of cccDNA.