The Mechanism Of HBV Hijacks Small Extracelluar Vesicles(sEVs) To Mediate Infection And Regulate Replication

OBJECTIVES:To establish the methodological basis of sEVs research,and explore the mechanism of HBV hijacks sEVs to mediate virus transmission and infection,promote HBV replication,and provide the theoretical basis for clinical diagnosis and treatment.METHODS:Firstly,ultracentrifugation was used to extract sEVs from the supernatants of HepG2 and HBV stable replicating cell line HepG2.2.15 cells,and electron microscopy,NTA,and WB identification.At the same time,DIO staining and tracing were used to determine the cell entry activity;CCK8,MTT,cell apoptosis test,and cell functional assays were used to determine the effects of paraformaldehyde-fixed DIO stained sEVs on cell viability and function,and to lay a foundation for research;Further,HepG2 cells were treated with sEVs derived from HepG2.2.15.The level of HBV DNA in the treated cells was determined by qPCR to confirm that sEVs mediate HBV transmission and infection;Finally,HepG2 cells were treated with miR-146a and FEN1 overexpressed or knocked down in sEVs derived from HepG2.2.15.qPCR,qRT-PCR and WB were used to determine HBV DNA,FEN1,miR-146a,and the downstream target gene IRAK1 and TRAF6,respectively,to analyze the specific molecular mechanism of sEVs regulating HBV replication.RESULTS:Firstly,transmission electron microscopy showed that sEVs derived from HepG2 and HepG2.2.15 cells had a typical bilayer vesicle-like structure;NTA counts of the two cells derived from sEVs particles,showed that more than 90%of the particle size was 144.40±14.70 nm,149.40± 10.40 nm,and their concentrations were(3.80±0.48)× 1012 particles/mL,(6.60±0.61)× 1012 particles/mL,respectively;WB identified sEVs of cell derived from sEVs sources with characteristic surface markers ALIX,CD9,and CD63;DIO fluorescence staining traced a large amount of fluorescent granular material in the cell pulp and perinuclear area;The positive rate of transfected cells which were transfected with paraformaldehyde-fixed DIO stained sEVs was 20.21±0.05%higher than paraformaldehyde non-fixed,and the cell apoptosis rate of the former was 12.65 ± 0.01%higher than the latter;Cell function intervention experiments have shown that the HBV DNA copy number was 3.33 times higher in paraformaldehyde-unfixed DIO stained HepG2.2.15 derived from sEVs transfected HepG2 cells than in the paraformaldehyde-fixed(P<0.05),and laying the foundation for relevant studies;Further,HBV DNA within sEVs of HepG2.2.15 origin was detected by qPCR,and HBV DNA was reduced by 5.17×104-fold compared to GW4869 after inhibition of sEVs(P<0.05);DIO fluorescence staining of HepG2.2.15 derived from sEVs before and after GW4869 inhibitor,transfected HepG2 cells were seen to have HBV-containing sEVs fully incorporated into the cytosol,and a large number of fluorescent particles accumulated in the cytoplasm,while there was no obvious fluorescent.particle material in the cytosol of the GW4869 intervention group;The corresponding cells were treated with HepG2.2.15 or HepG2 derived from sEVs,laser confocal spatial localization analysis of CD9 and CD63 dual fluorescent antibody-labeled sEVs reveals massive accumulation of sEVs in treated cells,and the positive rate of cell entry was 63.89%and 59.69%,respectively,qPCR detection of HBV DNA in HepG2.2.15 derived from sEVs treated with HepG2 cells showed a 6.4×104-fold reduction in HBV DNA in the transfected HepG2 cell group compared to that in the GW4869 post-sEVs inhibition group,indicating that HBV hosts sEVs to mediate transmission and infection;Finally,overexpression(or knockdown)of miR-146a and FEN 1 within HepG2.2.15 derived from sEVs treated with HepG2 cells,qRT-PCR detected a 1.63-fold and 1.49-fold increase(or 33.33-fold and 16.67-fold decrease)in miR-146a levels,and a 33.43-fold and 9.09-fold decrease(or 1.39-fold and 2.08-fold increase)in the levels of its downstream target genes IRAK1 and TRAF6,respectively,and a 1.79-fold and 1.42-fold increase(or 6.43-fold and 3.78-fold decrease)in FEN1 levels(P<0.05);HBV DNA copy number was increased 1.49-fold and 1.32-fold(or decreased 50.37-fold and 1.91-fold)by qPCR;FEN1 protein was increased 1.57-fold or 1.52(or decreased 2.07-fold and 2.31-fold)in transfected cells by WB,correspondingly,HepG2 derived from s.Vs treated with HepG2.2.15,the results also showed that sEVs enhance HBV DNA replication through internally contained miR--146a acts on downstream target genes IRAK1 and TRAF6 to affect FEN1,a key enzyme for HBV replication,to enhance HBV DNA replication.Conclusion:1.Extraction by ultracentrifugation,identification by transmission electron microscopy,WB and NTA detection,and tracirg of sEVs by DIO fluorescence staining can reliably extract and identify and differentiate sEVs,laying the foundation for relevant studies;2.Paraformaldehyde-fixed DIO stained sEVs tracer localization is better than paraformaldehyde-unfixed,and if the regulation of sEVs on cell activity and function are studied,paraformaldehyde-un flxed DIO-stained sEVs transfected cells are better;3.Hepatitis B virus hijacks sEVs mediate HBV transmission and infection;4.sEVs affect HBV replication key enzyme FEN1 by acting on downstream target genes IRAK1 and TRAF6 through miR-146a contained within them and thus enhance HBV DNA replicsation;5.Mutual promotion between miR-146a,a key factor for HBV replication carried by sEVs,and FEN1 positively regulate HBV replication.