ORIGINAL RESEARCH article
Front. Cell Dev. Biol.
Sec. Cancer Cell Biology
Proteome profiling of extracellular vesicles derived from Hepatitis B virus-infected hepatocellular carcinoma cell lines identifies PDCD11 as a carrier of viral RNAs
Provisionally accepted
- 1Institute of Post Graduate Medical Education And Research (IPGMER), Kolkata, India
- 2Indian Statistical Institute, Kolkata, India
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Introduction: Hepatocellular carcinoma (HCC) is a major outcome of chronic hepatitis B virus (HBV) infection. Molecular crosstalk among parenchymal, non-parenchymal, and immune cells in the liver tumor microenvironment (TME) is mediated through extracellular vesicles (EVs), which carry proteins, nucleic acids (DNA, coding/non-coding RNAs), lipids, and other molecules. This study investigates how HBV influences protein enrichment in EVs derived from HCC cells and vice versa. Methods: EVs were isolated from conditioned media of stable HBV-expressing HepG2.2.15 and HepG2-vector cells using a commercial kit. EV quality was verified using nanoparticle tracking analysis (NTA) and immunoblotting with EV-specific markers. Label-free LC-MS/MS proteomic analysis was conducted in triplicates and validated using HBV-infected Huh7 and HepG2 cells. Additional analyses included bioinformatics, transfection with full-length (FL) HBV plasmid, anti-sense oligo (ASO) treatment, qRT-PCR, immunoblotting, chromatin immunoprecipitation (ChIP), and functional assays. Student’s t-test was used for statistical analysis. Results: HBV infection led to a 3.4-fold increase in protein content in EVs from HepG2.2.15 cells (2293 proteins) versus HepG2-vector cells (677 proteins). Differential expression and validation with HBV-HCC liver tissue proteomics identified 103 commonly enriched proteins, mainly involved in DNA repair, RNA metabolism, and Golgi trafficking. Protein interaction network analysis revealed 10 hub proteins, including PDCD11, which facilitates the transfer of HBV RNA/DNA into EVs. PDCD11 depletion reduced HBV RNA (pre-genomic RNA, HBx, HBc, HBs mRNAs) and virion packaging into EVs. Intact FL-HBV genomes were detected in EV-associated viral core particles capable of infecting naïve hepatocytes. Further, transcription factor–miRNA–mRNA network analysis revealed that TFDP1 upregulates PDCD11 and other hub genes, while miR-1-3p represses them. TFDP1 binding to hub gene promoters was confirmed via ChIP-qPCR. ASO-mediated knockdown of TFDP1 or restoration of miR-1-3p suppressed proliferation, migration, EMT, and stemness in HBV-infected HCC cells. Conclusion: EV proteins from HBV-HCC cells represent potential therapeutic targets in the liver TME. Our findings highlight the therapeutic promise of TFDP1-ASO and miR-1-3p in managing HBV-driven HCC.
Keywords: Proteome, extracellular vesicles, EVS, Hepatitis B virus, HBV
Received: 09 Jun 2025; Accepted: 27 Oct 2025.
Copyright: © 2025 Dey, Das, Saha, Chowdhury, Datta and Banerjee. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Soma Banerjee, somabanerjee70@gmail.com
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