Targeted HBx gene editing by CRISPR/Cas9 system effectively reduces epithelial to mesenchymal transition and HBV replication in hepatoma cells

• Published in: Liver international 2024-02, Vol.44 (2), p.614-624
• Main Authors: Rawal, Preety, Tripathi, Dinesh Mani, Hemati, Hamed, Kumar, Jitendra, Tyagi, Purnima, Sarin, Shiv Kumar, Nain, Vikrant, Kaur, Savneet
• Format: Article
• Language: English
• Subjects: cancer stem cells (CSCs); Cell proliferation; Circular DNA; clustered regularly interspaced short palindromic repeats (CRISPR); CRISPOR; CRISPR; differentially expressed genes (DEGs); Editing; epithelial to mesenchymal transition (EMT); Genes; Genetic modification; HBV covalently closed circular DNA (cccDNA); Hepatitis B; Hepatitis B surface antigen; hepatitis B virus (HBV); Hepatocellular carcinoma; hepatocellular carcinoma (HCC); Hepatoma; Liver cancer; Pathogenesis; small guide RNA (sgRNA); Three dimensional models; three‐dimensional spheroids; Transcriptomics; Tumors
• ISSN: 1478-3223; 1478-3231
• DOI: 10.1111/liv.15805

Background and Aims Hepatitis B virus X protein (HBx) play a key role in pathogenesis of HBV‐induced hepatocellular carcinoma (HCC) by promoting epithelial to mesenchymal transition (EMT). In this study, we hypothesized that inhibition of HBx is an effective strategy to combat HCC. Methodology and Results We designed and synthesized novel HBx gene specific single guide RNA (sgRNA) with CRISPR/Cas9 system and studied its in vitro effects on tumour properties of HepG2‐2.15. Full length HBx gene was excised using HBx‐CRISPR that resulted in significant knockdown of HBx expression in hepatoma cells. HBx‐CRISPR also decreased levels of HBsAg and HBV cccDNA expression. A decreased expression of mesenchymal markers, proliferation and tumorigenic properties was observed in HBx‐CRISPR treated cells as compared to controls in both two‐ and three‐ dimensional (2D and 3D) tumour models. Transcriptomics data showed that out of 1159 differentially expressed genes in HBx‐CRISPR transfected cells as compared to controls, 70 genes were upregulated while 1089 genes associated with cell proliferation and EMT pathways were downregulated. Conclusion Thus, targeting of HBx by CRISPR/Cas9 gene editing system reduces covalently closed circular DNA (cccDNA) levels, HBsAg production and mesenchymal characteristics of HBV‐HCC cells. We envision inhibition of HBx by CRISPR as a novel therapeutic approach for HBV‐induced HCC.