Identification of Potential miRNA-mRNA Regulatory Axis of Intrahepatic cccDNA in Patients with Chronic Hepatitis B Virus Infection in the Grey Zone

Background: Intrahepatic covalently closed circular DNA (cccDNA) plays a critical role in the life cycle of the hepatitis B virus (HBV). Growing evidence suggests that microRNAs (miRNAs) may regulate cccDNA expression and contribute to the natural history of chronic hepatitis B (CHB). Objectives: This study aimed to investigate potential miRNA-mRNA regulatory axes of intrahepatic cccDNA in CHB-GZ patients and to identify new therapeutic targets. Methods: Thirteen CHB-GZ patients were included and divided into two groups based on cccDNA levels: Reference group (n = 7) with cccDNA 1 copy/cell and control group (n = 6) with cccDNA ≥ 1 copy/cell. Transcriptome-wide miRNA and mRNA expression profiles in liver tissue were determined. Differentially expressed miRNAs (DE-miRNAs) and mRNAs (DE-mRNAs) were defined by |logFC| log1.5 and P 0.05. Enrichment analyses of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were performed. Candidate miRNA-mRNA interaction pairs were acquired from miRTarBase, and a miRNA-mRNA network was constructed using Cytoscape based on the Spearman correlation coefficient (r). Results: We identified 19 DE-miRNAs and 340 DE-mRNAs. The most enriched GO terms were related to biological processes that regulate the virus life cycle, viral process, and viral genome replication. KEGG pathway enrichment analysis suggested that these predicted targets were associated with hepatitis B. Finally, we found a high correlation between miR-4295 and ZNF224, which suggests that they may form a potential regulatory axis of intrahepatic cccDNA in CHB-GZ patients. Conclusions: Our study suggests that miR-4295 and ZNF224 may be the potential regulatory axis of intrahepatic cccDNA in patients with CHB-GZ.