Gemcitabine Promote Transcription Of HBV PgRNA And HBV DNA Replication Of Hep G2.2.15 Cells And Its Molecular Mechanism

Objective:The incidence of hepatitis B virus infection and tumors in China is high,making the number of cancer patients with HBV infection numerous.However,some anti-tumor drugs on the market show reactivation activity for HBV,which adversely affects the conventional treatment of tumor patients infected with hepatitis B virus.At present,the HBV reactivationof various anti-tumor drugs has been reported,and the commonly used anti-tumor drug gemcitabine has recently been found to have the activity of reactivating HBV in clinical,but the molecular mechanism was unknown.To further explore the mechanism of anti-tumor drug reactivation of HBV,this study used HepG2.2.15 cells as a carrier to observe the effects of cell level and molecular level after stimulating with gemcitabine and analyzed the key signaling pathways and targets,in order to provide materials for mechanism of the anti-tumor drug which causing reactivation of HBV,and to lay a foundation for finding the commonality of these drugs.Methods:HepG2.2.15 cells which with the whole genome of hepatitis B virus transfected into were treated with different concentrations of gemcitabine?1?The cells were treated with 0,0.5,1,2,5,10,20,30,40,50,70,100?mol/L gemcitabine,and cell viability was measured by CCK8 at 24h,48h,and72h,respectively.?2?The cells were treated with 0,2,20,50,70,100?mol/L gemcitabine for 48h,the cell apoptosis and cell cycle were detected by flow cytometry.?3?The cells were treated with 0,2,20,50?mol/L gemcitabine for 24h,48h,72h and 96h.The expression of HBV pgRNA in cells was detected by real-time fluorescent quantitative PCR.?4?The cells were treated with 0,2,20,50,70,100?mol/L gemcitabine for 24h.The HBeAg in the cell culture supernatant were detected by ELISA.The HBV DNA in the supernatant was detected by real-time fluorescent quantitative PCR.?5?The cells were treated with 0,2,20,50,70,100?mol/L gemcitabine for 48h,and the mRNA expression levels of regulatory genes?hepatocyte nuclear factor 4?HNF4??,P38 mitogen-activated protein kinase?P38MAPK?,CAMP Responsive Element Binding Protein 1?CREB1?,nuclear factor-kappa B p65?NF-kbp65?,histonedeacetylase1?HDAC1?,peroxisome proliferator-activated receptor gamma coactivator 1-alpha?PGC1??,forkhead box protein O1?FOXO1?,sirtuin 1?SIRT1?,P53?which were related to the transcription of HBV pgRNA,HBV pgRNA itself were detected by real-time fluorescent quantitative PCR.These regulatory genes were divided into three major pathways based on cellular signaling pathways in previous studies and the interrelationship they regulated the transcriptional mechanisms of pgRNA:pathway 1,P38MAPK and its downstream transcription factors NF-kB,CREB1,and P53 pathways;pathway 2,transcription factors HNF4?,FOXO1,and transcriptional coactivators PGC-1?pathway;pathway 3,deacetylase HDAC1and SIRT1 pathways.The mean value of RNA from the latter concentration minused the mean value of the RNA from the previous concentration,getting the difference between HBV pgRNA and the difference of mRNA of each gene.Then the correlations between HBV pgRNA trend and each mRNA trend along with the trend of gemcitabine concentration were examined.?6?Cells were treated with 0,0.1,0.2,2,20,50,70 and 100?mol/L gemcitabine for 24 h,and the expression of protein HNF4?was detected by Western Blot.Results:?1?Gemcitabine inhibited the growth of HepG2.2.15 cells in a concentration-and dose-dependent manner within 72 h.The cell activity of 24h was about75-100%;the cell activity of 48h was about 30-65%;the cell activity of 72h was about 15-50%;and the cell activity decreased with increasing concen-tration at each time point.?2?Gemcitabine increased the apoptotic rate of HepG2.2.15 cells.The apoptotic rates of cells at concentrations of 2,20,50,70 and 100?mol/L were?2.90±0.92?%,?6.20±1.08?%,?7.87±,0.72?%,?6.10±0.57?%,and?4.73±0.51?%,respectively,compared with the control group?0.70±0.10?%,the increase was significant?P=0.014,0.001,0.003,0.043,and 0.000,respectively?;HepG2.2.15 cells were more arrested in the G₀G₁ phase because of gemcitabine,while less cells were at the S phase.The proportion of cells in the G₀G₁ phase treated by 2,20,50,70,100?mol/L gemcitabine were?72.31±3.77?%,?71.66±3.19?%,?73.63±5.51?%,?69.93±3.62?%,and?73.23±5.37?%,respectively,compared with the control group?39.36±2.86?%,the increase was significant?P=0.000,0.000,0.001,0.000,0.001,respectively?;the cells in the S phase treated by gemcitabine 2,20,50,70,100?mol/L gemcitabine took the proportion of?16.91±2.74?%,?16.49±5.58?%,?17.65±3.33?%?18.53±3.91?%and?18.59±1.91?%,compared withthe control group?49.37±2.38?%,the reduction was significant?P value=0.000,0.001,0.000,0.000,0.000,respectively?.?3?Gemcitabine significantly increased the HBV pgRNA in HepG2.2.15cells at the time of 24h,48h,and 72 h.Compared with the control group,HBV pgRNA increased in all groups treated by gemcitabine?P values were less than 0.05?,and showed significant time-and dose-dependent effects.Treated by Gemcitabine for 92h,HBV pgRNA also had an increasing trend.?4?The HBeAg ofcells which were treated by 2,20,50,70,100?mol/L gemcitabine at24hwere?14.90±2.07?,?23.70±0.10?,?17.30±2.82?,?23.23±1.20?,?16.07±1.79?S/CO,respectively,which were significantly increased compared with the control group?7.26±2.11?S/CO?P values were0.011,0.005,0.008,0.000,0.005?.?5?The correlation coefficients r of HNF4??P38MAPK?CREB1?NF-kb p65?HDAC1?PGC-1??FOXO1?SIRT1?P53 mRNA with HBV pgRNA were0.934,0.715,0.487,0.638,0.767,0.526,0.716,-0.231 and 0.434,respectively,The P values were 0.020,0.175,0.406,0.247,0.130,0.362,0.173,0.709 and0.465,respectively.Among all the three pathways,gemcitabine promoted the expression of HNF4?mRNA,and the trend of HNF4?mRNA was the highest correlated with the trend of HBV pgRNA.The transcription of HBV pgRNA is promoted by HNF4?,so gemcitabine promoted the transcription of HBV pgRNA and the replication of hepatitis B virus by promoting the expression of HNF4?.?6?Gemcitabine could increase the expression of HNF4?protein in HepG2.2.15 cells,and the results further supported the above mRNA levels results from protein levels.Colusion:Gemcitabine showed concentration and dose-dependent inhibition of HepG2.2.15 cell growth;gemcitabine increased the apoptosis of the cells;gemcitabine caused more cell arrest in G₀G₁ phase,and less in S phase;gemcitabine significantly increased HBV pgRNA in HepG2.2.15 cells and the increasing was time-and dose-dependent within 72 hours;HBeAg in cell culture supernatant of gemcitabine-treated cells at 24 h was increased;gemcitabine promoted HNF4?mRNA and HNF4?protein expression,and in all genes associated with HBV transcriptional regulation,HNF4?mRNA trends had the highest correlation with HBV pgRNA trends.Gemcitabine promoted HBV pgRNA transcription and HBV replication at the cellular level,and that was achieved by promoting the expression of the hepatitis B virus transcription factor HNF4?,not through other genes?P38MAPK,CREB1,NF-kb p65,HDAC1,PGC1?,FOXO1,SIRT1,P53?.