Regulation Of Hepatitis B Virus-encoded MiRNA On Viral Replication

[Objective] Hepatitis B virus(HBV)is an enveloped DNA virus with virions and subviral forms of particles that lack a core.The genomic material of HBV is circular,partially double-stranded DNA that is only 3.2 kb in size and is transcribed into four transcripts;i.e.,3.5 kb mRNA,2.4 kb mRNA,2.1 kb mRNA,and 0.7 kb mRNA.There are four overlapping open reading frames(ORFs): S,P,C and X,which encode hepatitis B viral surface antigen(HBs Ag),reverse DNA polymerase(HBV DNA Pol),core protein(HBc Ag,HBe Ag),and HBV X(HBx)proteins,respectively.micro RNAs(miRNAs)are a class of small single-stranded non-coding functional RNAs which regulate complementary mRNAs to play a biological function.More and more human and animal viruses have also been identified as encoding miRNAs.Viral miRNAs may profoundly affect host function and the natural progression of infection of some viruses in vivo.However,experimental evidence for HBV-encoded miRNA is lacking.Whether HBV-encoded non-coding RNA influences its own replication during infection remains a question.In the previous study,HBV-miR-3 was obtained from HBV-positive hepatocellular carcinoma(HCC)by deep sequencing.The aim of this study was to investigate the effect of HBV-miR-3 on HBV replication and its mechanism.[Methods] First,we predict the secondary structure of HBV-miR-3 through the biology information website,and analyze its conservatism in different HBV genotypes.HBV-miR-3 was confirmed by Northern blot and RT-q PCR.HBV-miR-3 was detected in HBV positive and negative HCC tissues,HBV positive and negative hepatocellular cell lines and non-hepatocellular cell lines by RT-q PCR asssy.Western blot assay was performed to investigate the generate process of HBV-miR-3.The expression of HBV-miR-3 in the RISC-Ago2 complex was studied by RNA immunoprecipitation and Western blot assay.Sucrose density gradient centrifugation and RNA immunoprecipitation assay to study how HBV-miR-3 is secreted by extracellular.Then the effect of HBV-miR-3 on HBV DNA and HBe Ag secretion was studied by ELISA.RT-q PCR assay was performed to study the effect of HBV-miR-3 on HBV DNA level,HBV complete viron,HBV pre-genomic RNA and total RNA. We investigated the target gene of HBV-miR-3 by Western blot,RT-q PCR and EGFP reporter assay,and further studied the target gene of HBV-miR-3,and further studied the target gene function.Finally,we investigated the effect of HBV-miR-3 and target gene on HBV promoter by luciferase assay.[Results] Hepatitis B virus encodes HBV-miR-3,HBV-miR-3 from the three transcripts of HBV 3.5 kb mRNA,2.4 kb mRNA,2.1 kb mRNA.HBV-miR-3 is present in HBV-positive hepatocellular carcinoma tissues and cells.The expression of HBV-miR-3 in serum of patients with HBV infection is higher than that of convalescence.The generate process of HBV-miR-3 is dependent on Drosha and Dicer's classic of the miRNA generation pathway,HBV-miR-3 exists in the RISC-Ago2 complex,mainly through the exosome and complete virion way to secrete to the extracellular.HBV-miR-3 inhibits HBs Ag,HBe Ag,HBV DNA level /HBV complete viron in supernatant,HBV replication intermediates,HBV pre-genomic RNA and total RNA expression.HBV-miR-3 targets HBV 3.5 kb transcripts to inhibit the expression of HBV core proteins,but has no effect on HBV DNA polymerase.HBV-miR-3 inhibits the activity of HBV Enh I / Xp by targeting host factor STAT3.[Conclusion] HBV encodes miRNAs,i.e.,HBV-miR-3.HBV-miR-3 inhibits HBV replication by down-regulation of HBV 3.5 kb transcripts,inhibiting HBc protein expression,and the other by targeting and downregulating host factor STAT3 to inhibit HBV Enh I / Xp promoter activity,thereby inhibiting HBV replication and transcription.