Mechanism For HBV PgRNA-Expressing Hepatocytes Promoting Liver Fibrosis By Activating Hepatic Stellate Cells Through Releasing Exosomes

Background&Aims:HBV pgRNA plays a crucial role in the Hepatitis B virus(HBV)life cycle,serving as a template for reverse transcription and translation of HBV polymerase and core antigen.The persistent replication of HBV leads to chronic inflammation,which is a significant contributor to liver cirrhosis and HCC.Serum and intrahepatic levels of pgRNA in patients with chronic hepatitis B(CHB)could reflect the extent of liver inflammation and fibrosis.Since HBV could only infect liver parenchymal cells via the sodium taurocholate cotransporting polypeptide(NTCP)receptor,pgRNA might indirectly participate in the chronic inflammatory-fibrotic process within the liver.Exosomes facilitate intercellular communication by transporting a diverse range of proteins,mRNAs,and non-coding RNAs.Moreover,exosomes exert regulatory effects on neighboring cells and modulate the immune microenvironment in the liver.They also play a role in regulating the activation of fibrosis effector cells,influencing the expression of fibrosis signaling molecules,and contributing to fibrosis development.In clinical practice,α-interferon serves as an efficacious antiviral drug against hepatitis B virus.It not only directly inhibits the production of hepatitis B virus protein,but also suppresses viral replication through immune response modulation and promotion of pgRNA degradation.The aim of our research is to elucidate the specific molecular mechanism underlying the promotion of liver fibrosis by pgRNA.By investigating the involvement of pgRNA in the process of liver fibrosis,we strive to provide a therapeutic direction for attenuating the progression from inflammation to liver cancer and reducing the incidence of hepatitis B-related liver cirrhosis.Additionally,our study aimed to identify effective molecular targets and treatments for clinical antiviral and anti-liver fibrosis interventions.Methods:1.Blood serum and liver tissues were collected from patients with hepatitis B-related liver cancer to investigate the correlation between pgRNA levels and liver fibrosis using qPCR and FISH experiments.2.The cell culture supernatant and the exosomes isolated from the supernatant were collected.The effects of these samples on the proliferation and migration ability and the expression level of activation marker of LX-2 cell lines were assessed using cytological and molecular experiments.3.To assess the impact of supernatant-extracted exosomes on the development of mice liver fibrosis,the histological techniques including HE staining,Sirius red staining,Masson staining,as well as immunohistochemical(IHC)staining for COL1A1 and α-SMA were used.4.Cytological and molecular experiments were performed on the LX-2 cell line following co-incubation with exosomes treated with Proteinase K or RNase A to elucidate the specific kind of factors influencing the activation state of LX-2 cells.5.Exosome microRNA sequencing was employed to identify pivotal molecules implicated in the progression of liver fibrosis.The differential expression of microRNAs was subsequently validated through nanoflow cytometry and qPCR techniques.6.The binding status between microRNA and pgRNA was confirmed through FISH assay,dual luciferase reporter assay,and RNA pull-down assay in vitro and in vivo.7.The uptake of extracellular vesicles containing microRNA by stellate cells was confirmed through FISH and qPCR experiments in vitro and in vivo.8.The relationship between the target microRNA and HSCs markers was verified through TCGA database data.The effect of the target microRNA on the proliferation and migration ability of LX-2 cell line was detected by cytological experiments.9.The impact of overexpressed microRNA molecules on mice liver fibrosis was assessed using HE staining,Sirius Red staining,Masson staining,and immunohistochemical staining for COL1A1 and α-SMA.10.The downstream target molecules of microRNA were predicted utilizing microRNA databases.The binding between the microRNA and downstream molecule was validated through dual-luciferase reporter assays and qPCR experiments.11.The effect of microRNA’s downstream target on the proliferation and migration ability of LX-2 cell line was detected by cytological experiments and the effect on the activation of the related signaling pathway were detected by molecular experiment.12.HepG2.2.15 cells were treated with alpha-interferon and the exosomes were extracted from the cell supernatant.The effect of interferon-treated cell-derived exosomes on LX-2 cell proliferation and migration were detected using cytological experiments and the activation of related signaling pathway were examined by molecular experiments.13.The impact of exosomes derived from interferon-treated liver cancer cells on mouse liver fibrosis was assessed using HE staining,Sirius Red staining,Masson staining,as well as IHC staining for COL1A1 and α-SMA.Results:1.The level of pgRNA in patient serum exhibited a positive correlation with the extent of liver fibrosis,while the level of pgRNA in patient liver tissue demonstrated a positive correlation with both the severity of liver fibrosis and the expression levels of ACTA2 and COL1A1,which served as markers for activated hepatic stellate cells.2.The supernatant and exosomes derived from pgRNA-overexpressing HepG2 cells could enhance the activation of LX-2 cell line,augment its proliferation and migration capabilities,as well as induce the expression of hepatic stellate cell activation markers.3.Injection of exosomes derived from pgRNA overexpressing cells led to elevated levels of hepatic inflammation,enhanced collagen fiber synthesis,and an increased abundance of COL1A1 and α-SMA positive cells,indicating a heightened degree of liver fibrosis.Conversely,administration of exosomes obtained from cells with suppressed pgRNA expression resulted in diminished liver inflammation,reduced collagen fiber production,and a decreased number of COL1A1 and α-SMA positive cells,suggesting a lower extent of liver fibrosis.4.Nucleic acids in exosomes extracted from cell supernatants could affect the activation state of the LX-2 cell line.5.Exosome microRNA sequencing revealed that the depletion of pgRNA resulted in alterations in the exosomal microRNA composition,with hsa-miR-148a-5p being identified as a prominent molecule.6.Hsa-miR-148a-5p could bind to pgRNA,and the expression of hsa-miR-148a-5p in exosomes decreased after binding.7.The LX-2 cell line could take up exosomes containing hsa-miR-148a-5p.8.There was a negative correlation between hsa-miR-148a and hepatic stellate cell markers ACTA2 and COL1A1 expression level.Moreover,the overexpression of hsa-miR-148a-5p effectively suppressed the proliferation and migration ability of LX-2 cells.9.After the overexpression of hsa-miR-148a-5p,there was a reduction in liver inflammation,decreased production of collagen fibers,a decrease in the number of COL1A1 and ACTA2 positive cells,which represented a lower degree of liver fibrosis in both CCl4-induced and TAA-induced liver fibrosis models.10.PDPK1 served as the downstream target of hsa-miR-148a-5p in LX-2 cell line,and hasmiR-148a-5p bound to the 3’-UTR of PDPK1 mRNA to inhibit its expression at the posttranscriptional level.11.PDPK1 enhanced the proliferative and migratory capacities of LX-2 cell line through the activation of PI3K-Akt signaling pathway.12.Exosomes derived from the supernatant of α-interferon-treated HepG2.2.15 cells exhibited inhibitory effects on the proliferation and migration ability of LX-2 cell lines through the activation of PI3K-Akt signaling pathway.13.Mice injected with exosomes derived from α-interferon-treated cells exhibited reduced hepatic inflammation,diminished collagen fiber synthesis,decreased presence of COL1A1 and α-SMA positive cells,and attenuated liver fibrosis in both CCl4-induced and TAAinduced liver fibrosis models.Conclusion:Our study has identified a positive correlation between the patient pgRNA level and degree of liver fibrosis in the clinical samples.The presence of pgRNA in liver parenchymal cells could modulate the activation state of non-parenchymal cells by binding to hsa-miR-148a5p,resulting in a reduction of hsa-miR-148a-5p assembled into exosomes by liver parenchymal cells.The HSCs ingested these exosomes,leading to a upregulation of PDPK1,which activated the PI3K-Akt signaling pathway to promote hepatic stellate cells activation.The decreased uptake of hsa-miR-148a-5p weakens its inhibitory effect on stellate cell activation,resulting in liver fibrosis formation.Further research has revealed that alphainterferon could inhibit the liver fibrosis process by suppressing pgRNA expression in parenchymal cells and downregulating the expression of PDPK1 and the activation level of PI3K-Akt signaling pathway in hepatic stellate cells,thus providing theoretical support for future clinical use of alpha-interferon in preventing and treating hepatitis B-related liver fibrosis.