Abstract
Background: Hepatitis B virus (HBV) affects around 250 million people worldwide, causing approximately 887,000 deaths annually, primarily owing to cirrhosis and hepatocellular carcinoma (HCC). The current approved treatments for chronic HBV infection, such as interferon and nucleos(t)ide analogs, have certain limitations as they cannot completely eradicate covalently closed circular DNA (cccDNA). Considering that HBV replication relies on host transcription factors, focusing on host factors in the HBV genome may provide insights into new therapeutic targets against HBV. Therefore, understanding the mechanisms underlying viral persistence and hepatocyte pathogenesis, along with the associated host factors, is crucial. In this study, we investigated novel therapeutic targets for HBV infection by identifying gene and pathway networks involved in HBV replication in primary human hepatocytes (PHHs). Importantly, our study utilized cultured primary hepatocytes, allowing transcriptomic profiling in a biologically relevant context and enabling the investigation of early HBV-mediated effects.
Methods: PHHs were infected with HBV virion particles derived from HepAD38 cells at 80 HBV genome equivalents per cell (Geq/cell). For transcriptomic sequencing, PHHs were harvested 1, 2-, 3-, 5-, and 7-days post-infection (dpi). After preparing the libraries, clustering and sequencing were conducted to generate RNA-sequencing data. This data was processed using Bioinformatics tools and software to analyze DEGs and obtain statistically significant results. Furthermore, qPCR was performed to validate the RNA-sequencing results, ensuring consistent findings.
Results: We observed significant alterations in the expression patterns of 149 genes from days 1-7 after HBV infection. The top 100 DEGs associated with mRNA metabolism, alternative splicing regulation, and spliceosomes were downregulated during HBV infection. Conversely, among the upregulated genes, significant changes were primarily related to endopeptidase inhibitor and UDP glucuronosyltransferase activity genes. We identified RNA-binding proteins involved in mRNA metabolism and alternative splicing regulation during HBV infection. We found RBM14 and RPL28 to be potential biomarkers for HBV-associated HCC.
Conclusions: Transcriptome analysis of gene expression changes during HBV infection in PHHs provided valuable insights into chronic HBV infection. Additionally, understanding the functional involvement of host factor networks in the molecular mechanisms of HBV replication and transcription may facilitate the development of novel strategies for HBV treatment.