Huh7, HepG2-Vector, and HepG2.2.15 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, HiMedia, #AL700A) containing 10% heat-inactivated FBS (ThermoFisher, #26140079), Pen-strep glutamine (100X, ThermoFisher Scientific, # 10378-016) at 37 °C in a humidified chamber containing 5% CO2. Huh7, HepG2 and HepG2.2.15 cells were gifted by Prof. Saumitra Das, Indian Institute of Science, Bangaluru; Prof. Partha Chakraborty, CSIR-Indian Institute of Chemical Biology, Kolkata and Prof. Shyam Kottili, University of Maryland, USA, respectively. HepG2.2.15 is constitutively HBV-producing cell line derived from HepG2 cells and widely used for the study of HBV infected HCC development (Hu et al., 2019).

The pre-miRNA clone of miR-1 in pRNAU6.1RNA/Neo vector (pPre-mir-1) was a kind gift from Prof. Raghunath Chatterjee, Indian Statistical Institute, Kolkata, India.

The full-length, linear monomeric HBV DNA of subgenotype-D1 was released from pJET1.2/blunt vector by digestion with 1U of SapI/µg at 37 °C for 12 h (Datta et al., 2018; Khatun et al., 2018), followed by gel purification using QIAquick gel extraction kit (Qiagen, # 28704). Huh7 (2 × 10⁵) cells seeded on 24-well plates were individually transfected with 200 ng of the HBV/D1-monomer using Lipofectamine 2000 (Thermo Fisher Scientific, # 11668019). The culture medium was replaced with fresh DMEM at 6 hours post-transfection. Cells were harvested after 48 h of transfection. 100 ng of pPremir-1 and TFDP1-anti-sense oligo (ASO) were transfected in Huh7 cells seeded in 24well and harvested after 48 h. Each experiment was set in triplicate and repeated twice.

Total RNA was isolated using RNAiso Plus (Takara, #9108), and about 2.5 µg of total RNA was used for cDNA synthesis for mRNA using the Revertaid cDNA synthesis kit [Thermo Fisher Scientific (TFS) #AB1453A] following the manufacturer’s protocols. qRT-PCR was performed with PowerUp™ SYBR™ Green PCR master mix (TFS) in QuantStudio7 (TFS), and analysis was performed as fold change in expression of genes using formula 2^−ΔCt, where ΔCt = (Ct Gene − Ct Internal control). Sequences of primers are presented in Supplementary Table S1.

Both HepG2-vector and HepG2.2.15 cells were cultured in 3.5 mL of DMEM with 10% exosome-depleted serum (Gibco, #A27208-03) for 72 h in a T25 flask. Media was collected and centrifuged for 20 min at 5000 RPM to pellet down the cellular debris and other contaminants. The supernatant was filtered with a 0.22 μm filter and subjected to EVs isolation using a kit from ExoCan Healthcare Technologies Pvt. Ltd., following the manufacturer’s protocol. Briefly, solution A (100μL/4 mL media) and solution B (2mL/4 mL media) were added, mixed, and centrifuged at 5,000 rpm for 1 h. EVs pellet was treated with RNaseA, DNase and Proteinase K sequentially to remove DNA, RNA and proteins from outside wall of EVs. EVs pellet (soluble protein free) was washed twice with 1 mL of PBS for 10 min, and the final pellet was suspended in 200 μL of filtered PBS for subsequent analysis.

Isolated EVs were subjected to protein extraction using ExoLyseP (ExoCan Healthcare Technologies Pvt. Ltd, India) following the manufacturer’s protocol. Briefly, 50 μL of lysis buffer (ExoLyseP) and 5 μL of protease inhibitor (25x) were added to resuspend the EV pellet. The mixture was incubated at 95 °C for 10 min and then chilled on ice for 5 min to lyse the EVs. The lysate was centrifuged at 8,000 rpm for 5 min, supernatant was collected and preserved at −80 °C freezer in small aliquots.

For immunoblot analysis, isolated proteins from EVs were quantified using Bradford reagent (Sigma, #B6916). About 25 μg of protein was boiled with 5xLaemmli buffer for 5 min before loading on the gel. Proteins were separated on 15% SDS-PAGE and transferred onto PVDF membrane (Amersham Biosciences, #GE10600023). After blocking with 5% skimmed milk, the membranes were incubated with primary anti-CD63 antibody (Novus, #NBP2,42225) and anti-Alix (Santa Cruz, #SC53538) at a 1:1,000 dilution overnight. It was then washed with TBS-T, and incubated with secondary antibody (Cell Signaling Technology #7076) at 1:5,000 dilution for 1 h at room temperature, washed, and developed using chemiluminescence kit (Pierce, #32106).

To determine the size and concentration of isolated EVs from the cell-cultured supernatant, freshly prepared EVs were subjected to NTA analysis using Malvern Nano Sight NS300 at the Indian Institute of Liver and Digestive Sciences (IILDS), Kolkata, India.

EV-protein was subjected to Liquid chromatography-mass spectrometry (LC-MS/MS) analysis from Sandor Proteomics Private Limited, Hyderabad. In brief, an equal amount of protein (100 μg) from respective EVs was diluted with 100 mM ammonium bicarbonate (NH₄HCO₃). The sample was briefly centrifuged, treated with 250 mM DTT, vortexed gently, and incubated at 95 °C for 1 h. Then, Iodoacetamide (250 mM) was added, briefly vortexed, and incubated in the dark at room temperature for 45 min. Trypsin was added and digested at 37 °C in a dry bath overnight. The resulting sample was vacuum dried and dissolved in 50 µL of 0.1% formic acid. After centrifugation at 10,000xg, the supernatant was collected into a separate tube.

The nanoscale LC separation of tryptic peptides was performed using an ACQUITY UPLC system (Waters Corp., USA). The separation of all samples was performed on an ACQUITY UPLC BEH C18 column (Waters, USA) (150 mm × 2.1 mm × 1.7 µm), an analytical reversed-phase column (Waters, USA). A 10 µL injection volume was used on a BEH C18 UPLC column for the separation of peptides. The samples were initially transferred to the pre-column using an aqueous 0.1% formic acid with a flow rate of 30 mL/min for 1 min. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. The peptides were separated using a gradient of 2%–80% mobile phase B for 45 min. The column was re-equilibrated to the initial conditions for 15 min. All samples were analyzed in triplicate.

The tryptic peptides were analyzed using a SYNAPT G2 HDMS™ mass spectrometer (Waters, Manchester, UK) with a hybrid quadrupole/ion mobility/orthogonal acceleration time-of-flight (OA-TOF) geometry.

The raw data acquired from the instrument was processed using Protein Lynx Global Server (PLGS) PLGS software 3.0.2, and each peptide sequence in FASTA format was matched against the UniProtKB/Swiss-Prot obliged sequences. The criteria considered for analysis were (i) peptide tolerance of 50 ppm, (ii) fragment tolerance of 100 ppm, and (iii) a minimum number of fragments match for peptides and proteins was 2 and 5, respectively. The minimum number of peptides matches for proteins was two. One missed cleavage site was allowed, and the fixed modification of carbamidomethyl-C and oxidation of M were specified. The identification of the protein was performed with a maximum 5% False discovery rate (FDR) in at least three technical replicate injections. For the identification and quantification of protein level, the observed intensity was normalized with the intensity of the identified peptides of the digested internal standard. Proteins with ≥ log₂fold change ±1 were considered, and protein tables generated by PLGS were merged. The raw file has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD056246.

Proteins identified in all three replicates of EVs-derived from HepG2.2.15 vs. HepG2-vector were only counted in this study. The data was validated using various public datasets such as (i) liver tissue proteomics data of HBV-HCC patients, and (ii) Exocarta, a collection of exosomal components (mRNA, lncRNA, miRNA, circRNA, proteins) used to validate enrichment of proteins.

Huh7 cells were transfected with HBV-monomer, and after 72 h, intracellular core particles were isolated to extract HBV-DNA as described earlier (Hu et al., 2019; Datta et al., 2018). HBV-DNA was quantified by real-time PCR and normalized with Renilla Luciferase readings. EV-encapsulated core particle was isolated by using the same method after digestion of EVs with DNase I (Roche) and RNase A (TFS) followed by inactivation.

Huh7 cells were transfected with desired plasmid in 24 well plate. After 48h, 1 × 10⁴ cells were added to the upper compartment of the 24-well Boyden chambers (8 µm pore, Corning) and 30% serum was added to the bottom chamber as chemo-attractant. The plate was incubated for 12–24 h at 37 °C and non-migrated cells were removed from the top while migrated cells to the lower surface of the membrane were fixed, stained with crystal violet, and counted under a microscope.

Following the same transfection protocol in Huh7 cells in 24 well plate, cells (1x10³/well) were seeded on ultra-low attachment (6-well) plates in serum-free DMEM/F12 medium supplemented with B27 (1×, TFS), EGF (20 ng/mL, TFS), and bFGF (20 ng/mL, TFS). Cultures were maintained at 37 °C in 5% CO₂, and spheroid formation was monitored for 7–10 days. Spheroids with a diameter ≥50 μm were imaged under a phase-contrast microscope and counted.

The promoter sequence of each gene was retrieved from UCSC database and −1,000 base pairs from transcription initiation site (+1) were analyzed using TFBIND web tool for searching of TFs TFDP1 was selected as common TF which with binds to all the seven genes.

After transfection with desired plasmids in 6-well plate for 48h, Huh7 cells (2.5× 10⁷) were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched with 125 mM glycine. Cells were spun down, added lysis buffer and sonicated to an average DNA fragment size of 200–500 bp. Chromatin was incubated overnight at 4 °C with 4 µg anti-TFDP1 antibody (Abclonal, #A5214) or with species-matched IgG as negative control, followed by captured with Protein A/G Sepharose beads. Beads were washed thrice and reverse-crosslinked at 65 °C. DNA was purified by using phenol/chloroform followed by ethanol precipitation and analyzed by qPCR using primers targeting the promoter/enhancer regions of interest. Data were normalized to input and presented as fold enrichment over IgG.

miRNet 2.0 was used to identify the miRNAs-mRNAs (hub genes) interaction. Subsequently, TCGA-LIHC miRNA expression data were used to verify the downregulation of miRNAs (adjusted p < 0.05, log2FC ≤ −0.5). The interaction network was visualized using Cytoscape, and miRNAs were ranked according to the number of targeted hub genes. Expression and survival analyses were performed using the “survival” and “survminer” R packages.

Targetscan tool was used to identify the target genes and its binding to the 3′UTR of the identified miRNA.

Statistical calculations were performed using Microsoft Excel and GraphPad PRISM 8 software (GraphPad Software, La Jolla, CA, USA). All data were expressed as mean with standard deviation. To evaluate the differences, the Student’s t-test was performed. P < 0.05 was considered as statistically significant.

EVs released from HepG2.2.15 and HepG2-vector cells cultured in Exo-free media were isolated using the kit from Exocan Private Limited. The quality and quantity of the EVs were estimated by immuno-blot analysis with anti-CD63, anti-Alix antibodies, and nanoparticle tracking analysis (Figures 1a,b).

To identify the cellular proteins enriched in the EVs of HBV-infected hepatocytes, total protein was isolated from EVs using kit and subjected to proteome analysis (LC-MS/MS) in triplicate. The proteome profiling revealed that a total of 2,293 and 677 proteins were retrieved from the EVs of HepG2.2.15 and HepG2-vector cells, respectively, implicating that the number of proteins released in the presence of HBV infection was 3.4-fold more than in the control cells. Further analysis revealed that 519 proteins were noticed to have differential expression (DE) (|Log₂FC|>1, p value <0.05) in HepG2.2.15-derived EVs compared to HepG2-vector cells. Among these 519 proteins, 512 proteins were accumulated more in the EVs of HepG2.2.15 cells (>2-fold), and 7 proteins were enriched in the EVs of HepG2-vector cells (>2-fold). Again, 432/512 proteins were explicitly enriched in the EVs of HepG2.2.15 cells, which were absent in the HepG2-vector cells-derived EVs (Figure 1c). The unsupervised hierarchical clustering heatmap represents the top 10% of the proteins in the EVs of HepG2.2.15 cells versus HepG2-vector cells (p < 0.05) (Figure 1d).

It is worthy to be noted that we have identified 28 EV-enriched host proteins which might be interacting with various HBV proteins (HBV-polymerase, HBx, HBcore, and HBs) and HBV-cccDNA as identified from published literature (Supplementary Table S2; Supplementary Figure S1).

Now, 512 DE proteins in the EVs-derived from HepG2.2.15 were subjected to Gene Ontology (GO) and REACTOME pathway enrichment analysis. GO analysis depicted the biological processes, cellular locations, and molecular functions of the EV-enriched proteins. Interestingly, biological processes revealed that these proteins were mostly contributed to protein depolymerization, RNA splicing, mRNA processing (UPF2, TCERG1, TBL3, PDCD11, CSTF3, DDX23, DHX16, LUC7L3, THOC2, THOC5) and DNA repair (LIG1, POLD1, EXO1, MLH1, MLH3, MSH3, MSH6) (Supplementary Figure S2). Thus, EVs were packaged with nuclear-exported and cytosol-enriched proteins. In-depth molecular function analysis with REACTOME also resonated with this result. In addition to RNA metabolism and DNA mismatch repair pathways, proteins functioning in membrane trafficking and vesicle-mediated transport (TMF1, RALGAPB, MYO5A, COG1, TRIP11, KIF21A, KIF1B, COPG2) pathways were also loaded in the EVs of HepG2.2.15 cells (Supplementary Figure S2).

Next, the enriched proteins in the EVs of HepG2.2.15 cells were compared with the protein profiles of tumor tissue from HBV-HCC (n = 101) versus adjacent tissue (n = 98) to confirm the influence of the DE proteins in the tumor microenvironment (Jiang et al., 2019). The data revealed that 103 EV-enriched proteins were also found to be overexpressed in the liver tissue of HBV-HCC patients (Figure 1e). To gain further insights, high degree hub proteins were determined with these 103 DE proteins using STRING and Cytoscape tools, with a medium confidence score 0.7 for the protein-protein interaction (PPI). The first-order PPI web created an extensive network comprising 63 nodes and 87 edges with an enrichment P value of 1.11 × 10⁻¹⁶ (Figure 2a). The node proteins were found to have a significant role in numerous regulatory pathways simultaneously and interact with multiple partners. Next, the maximal clique centrality (MCC) methods were used to select the hub genes from the PPI network using the CytoHubba plugin with default parameters in Cytoscape. The topological network parameters of MCC identified the highest-ranked 10 hub genes, namely, PRPF6, XAB2, DHX16, IK, RNF113A, POLD1, MSH6, PDCD11, MSH3, and UTP14A (Figure 2b).

Next, the expression of 10 hub genes were verified in the TCGA liver cancer dataset. A significant upregulation of all the 10 genes was observed in HCC compared to the adjacent normal tissue. Furthermore, Kaplan-Meier curve analysis and log-rank P values (<0.05) for 10 hub genes depicted that, except for MSH3, the expression level of 9 genes was significantly associated with the overall survival of HCC patients (P < 0.05) (Supplementary Figure S3a). To reinforce the HBV-specific hub gene expression, we reanalyzed the expression of these nine genes in HBV (n = 117) versus non-HBV samples (n = 254) of TCGA-LIHC. Four hub genes, namely, PRPF6, RNF113A, POLD1, and MSH6, were found to be significantly upregulated in HBV-infected samples (Supplementary Figure S3b). Thus, we validated the data with qRT-PCR using intracellular total RNA of HepG2.2.15 and HepG2-vector cells as well as Huh7 cells transfected with the monomer of FL-HBV genome compared to Huh7-vector, considering that the EV-enriched proteins were also overexpressed intracellularly. Five genes (PRPF6, DHX16, RNF113A, POLD1 and MSH6) were observed significantly upregulated in presence of HBV (p < 0.05) in both the cell lines while two genes (XAB2 and PDCD11) remained unchanged (Figures 3a,b).

Among all the hub genes, the EV-enrichment of an apoptosis-linked nuclear RNA binding protein (RBP), PDCD11, was further explored. First, we measured the intracellular and EV-enriched PDCD11 protein level in Huh7 and Huh7 transfected with HBV-monomer after treatment with scrambled oligo and PDCD11-ASO. The data was also validated in the EVs-derived from HepG2, HepG2.2.15 cells treated with scrambled oligo and PDCD11-ASO (Figure 3c). The data revealed that PDCD11 remained unchanged intracellularly but was significantly enriched in the EVs upon HBV infection as observed in both Huh7-HBV-monomer versus Huh7-vector cells and HepG2.2.15 versus HepG2-vector cells while PDCD11-ASO treatment depleted its intracellular and EV-level (Figure 3c). Interestingly, we also quantified intracellular HBV-specific RNAs such as pgRNA, HBx, HBc, and HBs and observed a reduction in their intracellular levels upon PDCD11-ASO challenge compared to control (Figure 3d), suggesting PDCD11 might regulate the expression of those viral RNAs. Thus, RIP assay was performed with anti-PDCD11 antibody using extract from Huh7 cells transfected with HBV-monomer and HBV-monomer + PDCD11-ASO. The qRT-PCR analysis with extracted RNA from RNA-RBP complex revealed that PDCD11-ASO treatment significantly reduced the interactions between PDCD11 and HBx/HBc mRNA, and EVs isolated from the same set of experiments depicted a reduction of HBx/HBc/HBs mRNAs (Figures 3e,f). Next, core particles were isolated from EVs-derived from Huh7 cells transfected with HBV-monomer in presence or absence of PDCD11 after digestion of EVs with RNase, DNase and Proteinase K to remove RNA, DNA and proteins from outside wall of the EVs. The Naïve Huh7 cells were incubated with these EVs for 48h, and isolated core particle-associated HBV-DNA and quantified HBV copy number. Though PDCD11-ASO treatment restricted HBV copy number in Huh7-transfected cells, no significant difference was noticed in naïve Huh7 cells treated with EVs (Figure 3g). This data suggests that EVs of HBV-infected cells might be enriched with infectious HBV-virions, which has the potential to infect naïve Huh7 cells and PDCD11 might be regulating this process. HBV-specific RNAs were also quantified after HBV-infected EV treatment in naïve Huh7 cells and similar results were obtained, i.e., HBx, HBc, HBs specific RNAs were dropped upon reduction of PDCD11 (Figure 3h). The mechanism behind overloading of PDCD11 in the EVs upon HBV infection and its role in persistent HBV infection requires further investigation.

Next, we investigated the transcriptional and post-transcriptional regulation on the expression of PDCD11. As we have identified 10 hub proteins in the EVs-derived from HBV-infected hepatocytes, we aimed to understand the mechanism of enrichment of these ten hub genes in the EVs of the Huh7+HBV and HepG2.2.15. We searched for a common TF for the ten hub genes and the ENCODE ChIP-seq data was analyzed. Five TFs, namely, TFDP1, SP1, NRF1, ZFX, and GTF2E2 were noted to activate the expression of more than 60% of the hub genes, and TFDP1 showed the highest occurrence (Figure 4a). The data was further verified using TFBIND tool and observed that TFDP1 has multiple binding sites on promoter of PDCD11, DHX16, PRPF6, MSH6, POLD1, RNF113A (Supplementary Figure S4). The expression of TFDP1 Figure 4b and other TFs was also validated using TCGA-LIHC data, and observed their higher expression in HCC compared to normal. Survival data also supported the fact that the high expression of TFDP1 was positively associated with poor prognosis of HCC over the period of 5 years (Supplementary Figure S5a). In vitro verification of TFDP1 expression disclosed that it was overexpressed in Huh7 cells in presence of HBV and TFDP1-ASO treatment suppressed it (Figure 4c). On the other hand, the expression of six hub genes was decreased upon TFDP1-ASO treatment in Huh7 cells transfected with HBV-monomer compared to scrambled ASO treated cells (Figure 4d). Furthermore, to confirm TFDP1 binding to the promoters of six hub genes such as PRPF6, RNF113A, POLD1, MSH6, PDCD11 and DHX16, ChIP analysis was conducted with anti-TFDP1 antibody and a significant enrichment of TFDP1 was noted in all the six promoters in presence of HBV compared to IgG (Figure 4e).

Next, miRNet2.0 was used to identify the list of miRNAs that could target these hub genes to understand the post-transcriptional regulation. The search criteria were restricted to Homo sapiens and only significantly downregulated miRNAs in HCC compared to normal from TCGA-LIHC data were considered. We obtained a total of 20 miRNAs in HCC which were targeting 10 hub genes. Subsequently, a network was constructed with the top 4 miRNAs and 10 hub genes which depicted that hsa-miR-1-3p alone could target 7/9 of the hub genes, while let7b, miR-124-3p, and miR-129-2-3p could target 5, 4, and 3 hub genes, respectively (Table 1; Supplementary Figure S5b). The expressions and survival analyses were performed with TCGA-LIHC datasets for these miRNAs and observed that all the four miRNAs were repressed in HCC compared to adjacent liver tissue. Restoration of each miRNA was associated with significantly improved survival of HCC patients (Supplementary Figure S5c). Here, we have validated the impact of hsa-miR-1-3p on the six hub genes, PRPF6, DHX16, POLD1, PDCD11, MSH6, and XAB2 after restoring its expression in HBV-monomer transfected Huh7 cells. The binding site of miR-1-3p to the 3′UTR of those six genes has been also verified from Targetscan database. A significant downregulation of the target genes was observed in presence of miR-1-3p compared to scramble miRNA and anti-miR-1-3p transfected cells revealed binding of miR-1-3p to the 3′-UTR or coding sequences of the genes (Figures 4f,g). Further functional analysis is needed to confirm the binding of miR-1-3p to the UTRs.

We have also investigated the impact of TFDP1 and miR-1-3p on HCC progression and observed that both depletion of TFDP1 and restoration of miR-1-3p suppressed cell proliferation and reduced expression of EMT (N-Cadherin, Vimentin) and stemness signatures (Oct4, Sox2 and Nanog) in HBV-monomer transfected Huh7 cells (Figures 5a–f). In addition, migration of Huh7 cells and number of spheroid formation in anchorage independent growth condition were significantly dropped upon treatment with TFDP1-ASO and premiR-1 while anti-miR-1-3p restored it in Huh7 cells (Figures 5g–j).

Thus, our findings highlight that HBV triggers accumulation of more proteins in the EVs-derived from infected hepatocytes than controls. HBV-DNA, HBV-RNAs and intact virions are also loaded into the EVs, and EVs-derived from HBV infected cells can infect naïve cells. One of the EV-enriched proteins, PDCD11, might serve as a carrier of HBV-RNA/DNA to the EVs. Most of the hub genes identified in the EVs are upregulated intracellularly by a common TF, TFDP1 while miR-1-3p suppresses these hub genes. This highlights the therapeutic potential of anti-TFDP1 and miR-1-3p for improved treatment in HBV-HCC patients (Figure 5).