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HNF4-α knockout reduces HEPG2 cell migration and mitochondrial membrane stability under hypoxic conditions

ABSTRACT: Hypoxia represents an essential component of the tumor microenvironment of hepatocellular carcinoma (HCC) and contributes to metabolic reprogramming and drug resistance. Understanding the molecular players underlying the development of hypoxia in HCC is essential. This work used CRISPR/Cas9 to knock out the hepatocyte nuclear factor 4-alpha (HNF4-α) and hypoxia-inducible factor 1-alpha (HIF1-α). We measured the resulting effects on proliferation, migration, drug sensitivity and mitochondrial membrane stability under hypoxic and normoxic conditions. Our data shows that knockout (KO) of HIF1-α, but not HNF4-α, sensitizes HEPG2 cells to doxorubicin under hypoxic conditions. Moreover, knockout of HIF1-α and HNF4-α increases HEPG2 migratory potential, independent of hypoxia. Furthermore, both HIF1-α and HNF4-α were necessary for the cells to respond properly to hypoxia and maintain mitochondrial function, as marked by the inability of HEPG2 to increase their mitochondrial membrane potential under hypoxic conditions upon HIF1-α and HNF4-α KO. Our data suggests that HNF4-α plays a regulatory role in cell migration and mitochondrial stability under hypoxic conditions in a preclinical model of HCC. This study aims to investigate the role of HNF4-α and HIF1-α genes in the cellular response to hypoxia in HepG2 liver cancer cells. By generating knockout (KO) clones for these two genes using CRISPR-Cas9 and subjecting them to intermittent hypoxia cycles, we aim to uncover the impact of these knockouts on hypoxia-related pathways. Genomic DNA amplification and Sanger sequencing were used to identify successful KO clones. Clones were then subjected to 15 cycles of intermittent hypoxia, alternating with recovery in normal aerobic conditions, and were compared with control clones grown under normal oxygen levels. The study includes both normoxia and hypoxia conditions to explore how these genes influence the cellular response to oxygen deprivation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE278597 | GEO | 2025/12/31

REPOSITORIES: GEO

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Project description:The high rates of mortality associated with epithelial ovarian cancer (EOC) are a direct consequence of its metastatic nature. Metastasis is dependent on many factors, among which activation of angiogenesis is most significant. Angiogenesis is, in turn, contingent upon the cellular response to hypoxia within the tumor microenvironment. Hypoxia-inducible factor 1 (HIF1) is a transcription factor composed of HIF1alpha and HIF1beta subunits and is the master regulator of the hypoxic response. It is therefore a critical mediator of tumor angiogenesis and metastasis. Regulation of HIF1 is primarily at the level of protein. In normoxia, the HIF1alpha subunit is hydroxylated via an oxygen- and iron-dependent mechanism and targeted for destruction. In hypoxia, low oxygen levels preclude hydroxylation and HIF1alpha is stabilized, allowing for its association with constitutively expressed HIF1beta to form bioactive HIF1. We have identified a novel mechanism of HIF1alpha regulation in EOC cells that involves microRNAs (miRs), ~22 nucleotide, non-coding RNA molecules that repress translation of target mRNAs by binding their 3’ untranslated regions (UTRs). Using microarray and qPCR analysis, we found that levels of miR-199a-1, a miR that is predicted in silico to target the HIF1alpha 3’ UTR, were reduced under hypoxia in EOC cells. We further demonstrated that miR-199a-1 directly targets the HIF1alpha 3’ UTR and overexpression of miR-199a-1 suppresses HIF1alpha protein levels and HIF1-driven gene expression. Moreover, cells stably overexpressing miR-199a-1 exhibit marked defects in migratory ability. These data were corroborated by our in vivo findings, which demonstrated that overexpression of miR-199a-1 causes significant reductions in tumor vessel density and tumor burden in nude mice. These findings provide insight into non-canonical, miR- and iron-based mechanisms of HIF1 regulation that may have important implications in the progression of EOC. miRNA expression analysis of A2780 epithelial ovarian cancer cells by microarrays

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HNF4-α knockout reduces HEPG2 cell migration and mitochondrial membrane stability under hypoxic conditions

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