Abstract
Background: Previous research has highlighted the roles of epidermal growth factor-like (EGFL) 7 and EGFL8 in hepatocellular carcinoma (HCC), while EGFL9 has been implicated in other cancers such as breast cancer. Despite this, its role in HCC remains unexplored. This study aims to investigate EGFL9's impact on HCC progression. Methods: EGFL9 expression was evaluated in 30 HCC tissue samples and 5 HCC cell lines with different metastatic potentials using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Serum EGFL9 levels were measured in 30 HCC patients via ELISA. EGFL9 knockdown and overexpression HCC cell lines were established, and a series of assays including MTT, apoptosis, wound healing, Transwell, subcutaneous tumor formation, and tail vein injection metastasis experiments in nude mice were performed. To investigate the underlying mechanisms, transcriptomics, proteomics, and metabolomics analyses were conducted on the Huh-7 HCC cell line. Western blot and function rescue experiments were carried out to validate signaling pathways and metabolic reprogramming induced by EGFL9. Results: EGFL9 expression was significantly upregulated in both HCC tissues and cell lines, with higher expression correlating with HCC progression. Elevated serum EGFL9 levels were observed in HCC patients, suggesting its potential as a diagnostic marker. Both in vitro and in vivo experiments demonstrated that EGFL9 knockdown inhibited HCC cell proliferation, invasion, survival, and metastasis. Transcriptomic and proteomic analyses revealed that EGFL9's function may be linked to the activation of the PI3K-AKT signaling pathway, which was further validated through inhibition experiments targeting EGFR and AKT.