Study On The Mechanism Of MiR-574-5p In Exosomes Secreted By Macrophages To Inhibit HBV Replication In Liver Cells Under The Effect Of Interferon

【Backgrounds】Approximately 290 million people worldwide are infected with hepatitis B virus（HBV）^[1].Among untreated patients with hepatitis B,the cumulative incidence of cirrhosis after 8 years is as high as 8%-20%,and the incidence of liver cancer after 5 years reaches2%—5%^[2].The"clinical cure"（sometimes called"immunological cure"）of chronic hepatitis B refers to the loss of hepatitis B surface antigen（HBsAg）in the patient’s serum,with or without seroconversion of HBsAg,and HBV DNA is consistently below the lower limit of detection.The definition of"viral cure"in virology is the complete elimination of hepatitis B virus,including HBsAg,HBV DNA,and HBV ccc DNA in liver cells^[3].Peginterferon alfa（Peg IFN-α）can greatly extend the metabolism time of interferonα（Interferon alfa,IFN-α）,which is a commonly used drug for the treatment of chronic hepatitis B patients.Several clinical experiments have shown that Peg IFN-αcan significantly increase the rate of HBsAg loss in CHB patients^[4],and can reduce the level of HBV ccc DNA in liver cells^[5].In-depth study of the mechanism has great significance for new strategies for the treatment of CHB patients.Exosomes are a class of small vesicles that exist outside the cell.They have been found to play an important role in exchanging and transmitting information between cells or tissues and organs in many diseases^[6].For example,exosomes can participate in the transmission of HBV-related components and affect NK cell function^[7],in addition,exosomes can mediate the antiviral effect of IFN-α^[8].Kuffer cells are macrophages in the liver and have an inflammatory response under the stimulation of lipopolysaccharides,and play an important role in the process of anti-viral infection and the immune response of tumorigenesis^[9-11].Studies have shown that exosomes can mediate the antiviral effect of IFN-α-treated liver non-parenchymal cells（including Kupffer cells）^[8].Micro RNA（microRNA,miRNA）is a type of single-stranded nucleic acid with a length of 19-25nt.It can bind to the UTR region of the target gene and regulate the expression of the target gene^[12].Many miRNAs can directly or indirectly bind HBV-related m RNAs to regulate their translation and expression,including HBV polymerase m RNA and pregenomic RNA（pg RNA）,thereby affecting the stability of HBV genes^[13].In addition,as a"carrier"in exosomes,miRNAs can be exchanged between cells to further"remotely"regulate gene expression in cells.This remote regulation can even affect the transcription or replication of viruses in cells,including HBV^[7],HCV^[14],HIV^[15],EBV^[16]and other viruses.Although some studies have reported that exosomes can mediate the antiviral effects of interferon,little is known about whether miRNAs in exosomes will play an effect in this process and the specific mechanisms involved.Therefore,our research aims to reveal whether macrophage exosomes have anti-HBV replication effects and the molecular mechanism of exosomes exerting antiviral effects.【Methods】Serum specimens from patients with good response to Peg IFN-αwere selected.In this study,the group with good response（also called the response group）was defined as the quantitative decrease of HBsAg by more than 0.5 log ₁₀ IU/ml from the beginning of interferon treatment（ie,baseline）to 12 weeks;otherwise it was the group with non-response（non-response group）.The exosomes were extracted from the serum and identified by means of electron microscopy,Western Blot（WB）,and nanoparticle tracking analysis（NTA）.The above-mentioned serum exosomes were added to the Hep AD38 cell culture system,and the contents of each virus component in the supernatant and cells were detected,including the supernatant HBsAg,hepatitis B antigen（HBe Ag）quantification,HBV DNA quantification,and Intracellular HBV ccc DNA levels were used to observe the effects of serum exosomes on liver cell HBV replication in patients at different periods after interferon treatment.The total RNA of the exosomes in the patient’s serum was extracted,the miRNA content was detected by deep sequencing technology,and the differentially expressed miRNA in the patient’s exosomes was verified by RT-q PCR.In the in vitro cell experiment,the exosomes in the supernatant of the macrophage cell line after IFN-αtreatment were extracted and identified,and they were applied to liver cells to observe the anti-HBV ability of the exosomes.RT-q PCR was used to detect miRNA expression in exosomes.By comparing the differentially expressed miRNAs from serum exosomes of patients and THP-1-derived macrophage exosomes after treated with interferon,three exosome miRNAs were significantly increased.Then,observe the antiviral ability of these three miRNAs separately,and predict the possible binding sites of the three miRNAs and HBV by means of bioinformatics technology such as miRbase and RNA22 database,and construct the plasmids and miRNA precursors plasmids related to the luciferase verification experiment.Luciferase validation experiment was performed to verify the accuracy of miRNA binding to the predicted site.【Results】Serum exosomes from patients who responded to Peg IFN-αtherapy at 12 weeks had greater ability to resist HBV replication than baseline exosomes,including the ability to reduce HBsAg,HBe Ag,HBV DNA in the supernatant and HBV ccc DNA levels inside the cell of HBV replicated cell lines.Deep sequencing of miRNAs in patient serum exosomes and subsequent in vitro validation experiments of patient serum exosomes showed that after treatment with interferon,the expressions of miR-193a-5p,miR-25-5p,and miR-574-5p were all up-regulated both from patient serum exosomes and THP-1 macrophage exosomes.Combining mimics and inhibitors of these three miRNAs,it was found that some miRNAs have inhibitory effects on virological indicators of HBV cell lines,and some of the miRNA inhibitors can restore the anti-HBV effect of macrophage exosomes after IFN-αtreatment.This indicates that miRNAs are involved in the antiviral process of macrophage supernatant exosomes.Further combined with the bioinformatics analysis database（RNA22,miRbase database）,it was found that miR-25-5p and miR-574-5p could potentially be combined with HBV m RNA sites（3087-3093 and 2750-2757）.Luciferase reporter plasmids（containing a predicted site sequence or a mutant sequence）of the HBV-related site were further constructed,and precursor miRNA（precursor miRNA,pre-miRNA）plasmids and renilla plasmid were used as controls.The luciferase report experiment showed that miR-574-5p can reduce the luciferase activity of plasmids containing HBV predicted sites,but not the mutant sequences,indicating that miR-574-5p can specifically bind to the corresponding site of HBV pg RNA,thereby exerting anti-HBV replication effect.However,using miR-25-5p,no change in luciferase activity containing the corresponding site of HBV pg RNA.This shows that miR-574-5p can bind to the corresponding site of HBV pg RNA（nt2750-2757）,which overlapping with the pol m RNA of HBV gene.Studies have found that this miRNA can also bind to pol m RNA,which promotes the degradation of polymerase.【Conclusion】Our studies confirmed that after interferon treatment,macrophage exosomes can carry miRNA（mainly miR-574-5p）,enter HBV-infected liver cells,and combine with HBV pg RNA to inhibit HBV transcription and replication.