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HNF4-α Knockout Promotes HepG2 Cell Migration and Reduces Mitochondrial Membrane Potential under Hypoxic Conditions
Tareq Saleh1, Mohammad A Ismail2,3, Mirvat Surakhy4
1Department of Pharmacology and Public Health, Faculty of Medicine, The Hashemite University, Zarqa, 13133, Jordan.
Introduction/Objective:
Hypoxia is a critical component of the tumor microenvironment contributing to metabolic reprogramming and drug resistance. Subsequently, elucidating the molecular mechanisms involved in hypoxia regulation in tumor cell models is essential.
Methods:
This study used CRISPR/Cas9 to knock out hepatocyte nuclear factor 4-alpha (HNF4-α) and hypoxia-inducible factor 1-alpha (HIF1-α) in HEPG2 cells. This study then assessed the effects of both knockouts (KO) on proliferation, migration, drug sensitivity, mitochondrial membrane stability, and gene expression in HEPG2 cells under both hypoxic and normoxic conditions.
Results:
Our data demonstrate that HIF1-α KO, but not HNF4-α KO, sensitizes HEPG2 cells to doxorubicin under hypoxic conditions. Additionally, the KO of both HIF1-α and HNF4-α enhances cell migration, irrespective of oxygen levels. Importantly, both HIF1-α and HNF4-α are required for cellular response to hypoxia and the maintenance of mitochondrial function, as evidenced by the loss of mitochondrial membrane potential increase under hypoxia in KO cells. Microarray-based transcriptomic profiling of HEPG2 cells revealed separation between HNF4-α and HIF1-α KO cells and controls under hypoxic versus normoxic conditions, with a markedly higher number of differentially expressed genes in hypoxia (up to 5,595 genes; adjusted p < 0.05, log2FC ±1.5). Both HNF4-α and HIF1-α contribute to hypoxia-responsive pathways, with impaired induction of key genes such as VEGFA and AK4 in KO cells, while GSEA indicated minimal effects of HNF4-α loss under hypoxia but pronounced metabolic pathway disruption in HIF1-α KO cells. Moreover, key regulators of mitochondrial function and mitophagy, including FUNDC1, MIEF1, and MIEF2, were upregulated in control hypoxic cells but not in KO cells.
Discussion:
These findings suggest that, in this single HEPG2 cell line model, HNF4-α contributes to the regulation of cell migration and mitochondrial stability under hypoxic conditions.
Conclusion:
Further investigation into the role of HNF4-α in tumor biology is encouraged.
