Over Expression Of NR6A1 Inhibits HBV Replication And Transcription

Objective: To screen the genes of the nuclear receptor family that affect the transcriptional activities of HBV enhancers or core promoter,and to characterize the role of the initially screened gene germ cell nuclear factor(NR6A1)on the transcription and replication of HBV.Methods:(1)cDNA was obtained by reverse transcribe the mRNA from HepG2 cells.Different nuclear receptor genes were amplified from the cDNA and cloned into the plasmid PCH9.(2)the plasmid pcore-Rluc with Renilla luciferase reporter gene was successfully constructed.(3)the nuclear receptor gene expression plasmids were co-transfected with the plasmid pcore-Rluc respectively.The luciferase activity was assayed to screen the nuclear receptor genes that have an effect on the transcriptional activities of the enhancers or the core promoter.(4)The core promoter or enhancer deletion mutants were constructed,and these constructs were cotransfected with NR6A1 respectively to identify the sequence interacting with it.(5)The plasmid of HBV1.3 and NR6A1 were co-transfected into HepG2 cells.Western blotting was used to confirm the expression of NR6A1 protein.The intracellular HBV replication intermediates were analyzed by Southern blot,and HBV RNA was assayed by Northern blot.(6)The NR6A1 truncated variants was constructed.The key regions of(7)NR6A1 for its activity were characterized by co-transfection experiments.Results:(1)Eight nuclear receptor genes were successfully amplified and cloned.(2)Through the co-transfection of the nuclear receptor gene expression plasmids and the luciferase reporter plasmid pcore-Rluc,the gene NR6A1,which has an obvious influence on the luciferase activity of HBV enhancer / core promoter,was screened out.(3)NR6A1 significantly inhibited the activity of HBV enhancer or core promoter,with the Rluc activity decreased about 5 times in the experimental group(0.198±0.009)compared with the control group(1.000±0.319)(q=31.19,P=0.000).(4)The truncated mutation plasmids of Pcore-Rluc PCH9-1744-Rluc(BCP),PCH9-1636-Rluc(BCP+Enh II)and PCH9-1635/1747-Rluc(BCP+Enh I)were successfully constructed.(5)The truncated mutation plasmids of Pcore-Rluc were co transfected with NR6A1.By detecting the activity of luciferase,it was found that the luciferase activity of the experimental group(NR6A1 and PCH9-1635/1747-Rluc co-transfection)had no significant changes(t=1.018,P=0.367)compared with the control group(vector and PCH9-1635/1747-Rluc co-transfection).The relative luciferase activity of the experimental group(NR6A1 and the PCH9-1744-Rluc co transfection)was 0.504±0.023,which was 50% lower than the control group(1.000±0.099(t=6.534,P=0.0226)),while the relative luciferase activity of the experimental group(NR6A1 and PCH9-1636-Rluc)was 0.089±0.002,which was 92% lower than the control group(1.000±0.042(t=31.59,P=0.001)).(6)The expression plasmid of 3×flag-NR6A1 was successfully constructed,and the expressionof 3×flag-NR6A1 protein was confirmed by Western blot after cotransfection of 3× flag-NR6A1 expression plasmid with HBV1.3.The level of HBV DNA replication in the experimental group(HBV1.3 and 3× flagNR6A1 co transfection)was significantly lower than that of the control group(HBV1.3 and vector co-transfection).The level of HBV RNA in the experimental group was significantly lower than that of the control group(P=0.004).(7)The truncated mutant plasmids of NR6A1NR6A1 DBD,NR6A1LBD,NR6A1 N and NR6A1 C were successfully constructed.(8)There was no significant change between the control group(vector and pcore-Rluc co-transfection)(10.228)and the experiment group(NR6A1DBD+pcore-Rluc(1.011±0.070)and NR6A1 LBD+pcore-Rluc(0.900±0.017)).The relative value of luciferase activity the experiments group(NR6A1N +pcore-Rluc or NR6A1C+pcore-Rluc)was not different from that of control group(NR6A1 and pcore-Rluc cotransfection)(1±0.170(P > 0.05)).The relative value of luciferase activity the experiments group(NR6A1C+pcore-Rluc(1.160±0.078(P >0.05))was not different from the control group(NR6A1 and pcore-Rluc co transfection)(1.150±0.141).Conclusion:(1)Eight nuclear receptor family members,NR6A1 inhibits HBV core promoter and enhancer II activity significantly.(2)Over expression of NR6A1 can inhibit the transcription and replication of HBV.(3)Core promoter and enhancer II are the key parts of Pcore-Rluc inhibiting the transcription and replication of HBV.(4)The DNA binding domain(DBD)and ligand binding domain(LBD)of NR6A1 can not inhibit the transcription and replication of HBV alone,and the existence ofthe hinges can be used to inhibit the activity of the core promoter and enhancer of HBV.Over expression of NR6A1 inhibited the transcription and replication of hepatitis B virus in HepG2 cells.