Zona pellucida glycoprotein 3 (ZP3) is an important member of the zona pellucida glycoprotein family. However, the underlying mechanisms and immune regulation in hepatocellular carcinoma (HCC) remain unclear.

The protein expression level of ZP3 in HCC tissues was detected via Western blotting (WB) and immunohistochemistry (IHC). The impact of ZP3 on tumor microenvironment (TME) modulation in HCC was analyzed via the TCGA database, and its potential for immunotherapy response was assessed via the Tumor Immune Dysfunction and Exclusion (TIDE) database. Lentivirus-mediated knockdown and overexpression of ZP3 were performed in HCC cell lines, both in vitro and in vivo via various techniques, including immunofluorescence (IF), EdU, wound healing, colony formation, migration, transcriptome sequencing, flow cytometry, and xenograft assays.

We confirmed that ZP3 protein levels were significantly greater in HCC tissues than in normal tissues through WB and IHC analyses. Functionally, ZP3 knockdown in Huh7 cells reduced proliferation, migration, and invasion and slowed tumor growth in xenograft models; conversely, ZP3 overexpression in LM3 cells intensified these oncogenic traits. Mechanistically, enrichment analysis of the transcriptome data suggested that ZP3 might promote HCC progression via the Notch signaling pathway. Furthermore, ZP3 knockdown decreased key components of the Notch pathway (Jagged1, Notch1, NICD1, and Hes1), whereas ZP3 overexpression activated this pathway. ZP3 expression was significantly correlated with tumor-infiltrating immune cells (such as M0 macrophages, T cells, and NK cells) and TME scores in HCC. Notably, patients with high ZP3 levels presented lower TIDE scores, indicating reduced immune evasion potential and a better response to immunotherapy.

ZP3 promotes HCC progression by activating the Notch pathway and modulating the TME, highlighting its potential as both a prognostic biomarker and a therapeutic target.