Ananya Ajith1, Françoise Smets1, Etienne Sokal1, Mustapha Najimi1 1Laboratory of Pediatric Hepatology and Cell Therapy (PEDI), Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, Brussels, Belgium Email : ananya.ajith@uclouvain.be Background and Aims: Chronic liver disease (CLD) progression from fibrosis to hepatocellular carcinoma (HCC) involves profound immune dysregulation, with Kupffer cells (KCs) playing a pivotal role. KCs are liver resident macrophages that form the primary line of defence in liver. An imbalance in the hepatic macrophage population becomes evident during CLD progression from liver fibrosis to HCC. This study aims to explore the deregulated (DE) miRNA and mRNA profiles in mouse KCs to elucidate key molecular mechanisms driving liver fibrosis and HCC. Method C3H mice were given a single injection of diethylnitrosamine (DEN) at 4 weeks of age, followed by continuous injections of carbon tetrachloride (CCl4) for 6 and 21 weeks to induce liver fibrosis and HCC. KCs were isolated by fluorescence-activated cell sorting (FACS) from the non-parenchymal cell fraction of enzymatically digested liver (Collagenase P- Roche), and total RNA was extracted using miRNeasy micro kit (Qiagen), for sequencing. Differential expression and functional enrichment analyses were performed to identify key pathways and biological processes. Results: For both miRNA and mRNA sequencing, the fold change and p value threshold were set at | logFC | > 1 and p value <0.05. In-depth miRNA sequencing identified 7 DE-miRNAs in fibrosis and 15 in HCC compared to naïve controls. Among the miRNAs analyzed, mmu-miR-210-3p, was uniquely expressed in fibrosis and mmu-miR-126a-5p, mmu-miR-3082-5p, mmu-miR-199a-5p, mmu-miR-125b-5p, mmu-miR-29c-3p, mmu-miR-5128, mmu-miR-714, mmu-miR-709, mmu-miR-362-3p expressed only in HCC, compared to the naïve liver. Notably, mmu-miR-199b-5p was upregulated 4-fold in both conditions. Transcriptomic analysis revealed 140 downregulated and 686 upregulated DEGs in fibrosis, and 243 downregulated and 1,068 upregulated DEGs in HCC compared to naïve controls, and 56 unique to HCC compared to fibrosis. Gene set enrichment analysis highlighted dysregulation of pathways related to immune response, cytokine-chemokine signalling, and inflammatory processes in both fibrosis and HCC groups. DE-genes of the cytokine pathways include Ccr2, Irf4, Il20rb in fibrosis and Cxcl13, ccl12 in HCC. Inflammatory genes include Ednrb, Il33, Il2ra in fibrosis and CCl5, Ilr12 in HCC. Conclusion: Our study identified distinct DE-miRNAs and DE-genes in KCs during liver fibrosis and HCC, compared to naïve group. These findings highlight the critical role of KCs driving inflammation and immune modulations during CLD progression to tumorigenesis, offering potential therapeutic targets.
Ajith et al. (Wed,) studied this question.