HNF-4α regulates hepatoma cell growth and apoptosis through the IGFBP-2/Notch1 signaling pathway

Abstract: Background: Hepatocyte nuclear factor 4α (HNF-4α) is a liver-enriched transcription factor that is downregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms by which it regulates the malignant phenotype of HCC cells remain unclear. Objectives: To investigate whether HNF-4α affects HCC cell proliferation, invasion and apoptosis by regulating the IGFBP-2/Notch1 signaling pathway. Methods: HNF-4α was overexpressed and silenced in HepG2 cells using a pcDNA3.1-HNF-4α plasmid and HNF-4α-specific siRNA, respectively. RT-qPCR and Western blotting verified transfection efficiency. Cell proliferation and invasion were assessed by MTT and Transwell assays, respectively. Apoptosis was evaluated by Caspase 3 activity assay. IGFBP-2 secretion was measured by ELISA. Protein expression of Bcl-2, Bax, IGFBP-2 and Notch1 was detected by Western blotting. Results: HNF-4α overexpression significantly inhibited HepG2 cell proliferation (by approximately 35% at 48 h and 45% at 72 h) and invasion (by approximately 55%), while markedly increasing Caspase 3 activity (approximately 2.8-fold) and the Bax/Bcl-2 ratio (from 0.42 to 2.18). Moreover, HNF-4α overexpression significantly suppressed IGFBP-2 secretion (by approximately 60%) and downregulated IGFBP-2 (by approximately 55%) and Notch1 NICD (by approximately 50%) protein expression (all P < 0.001). Conversely, HNF-4α knockdown produced opposite effects, significantly promoting proliferation (by approximately 40% at 48 h and 55% at 72 h) and invasion (by approximately 70%), decreasing Caspase 3 activity (by approximately 55%) and the Bax/Bcl-2 ratio (from 0.45 to 0.19) and upregulating IGFBP-2 (approximately 2.2-fold) and Notch1 NICD (approximately 1.8-fold) expression (all P < 0.01). Conclusion: HNF-4α inhibits HCC cell proliferation and invasion and promotes apoptosis through negative regulation of the IGFBP-2/Notch1 signaling pathway, providing new experimental evidence for the tumor-suppressive mechanism of HNF-4α in HCC.