Hepatocellular carcinoma (HCC) is a highly lethal malignancy driven by both intrinsic oncogenic pathways and immune microenvironmental regulation. Emerging evidence suggests that DNASE1L3 may influence tumor biology and immune responses; however, its specific roles in HCC progression and macrophage-mediated regulation remain unclear. This study aimed to elucidate the biological functions of DNASE1L3 in HCC and to determine how it modulates tumor behavior and immune interactions.

Bioinformatics analyses of the GSE41804 and Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) datasets were used to identify hub genes. Functional assays assessed the impact of DNASE1L3 on HCC cell proliferation, migration, invasion, and cell cycle progression. The effects of DNASE1L3 on macrophage polarization and the Wnt/β-catenin signaling pathway were examined using a co-culture system. An HCC organoid model was established to further validate its regulatory function.

Eight prognostic signature genes were identified, with deoxyribonuclease I-like 3 (DNase I-like 3) selected as the hub gene. DNASE1L3 overexpression suppressed HCC cell growth, inhibited migration and invasion, induced G1 arrest, and modulated epithelial-mesenchymal transition (EMT) markers. DNASE1L3 knockdown promoted M2-like macrophage polarization. Mechanistically, DNASE1L3 interacted with β-catenin to enhance its ubiquitination and degradation, thereby inhibiting Wnt/β-catenin signaling and reducing PD-L1 expression. DNASE1L3 overexpression similarly restricted organoid growth and suppressed pathway activity.

DNASE1L3 acts as a negative regulator of HCC progression by targeting the Wnt/β-catenin pathway and reducing PD-L1 expression, thereby influencing both tumor cell behavior and macrophage-mediated immune responses.