Effects of HIF-1α Knockdown on Cell Viability and Oxidative Stress in Oxygen–Glucose-Deprived Human Brain Microvascular Endothelial Cells
Year: 2026
Type: preprint
Abstract: Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of cellular responses to hypoxia. This study examined the effects of HIF-1α knockdown on cell viability and oxidative stress in human brain microvascular endothelial cells and explored a putative HIF1A-AS2/miR-27a-3p/HIF1A regulatory relationship. Public stroke-related transcriptomic datasets were analyzed, and hCMEC/D3 cells were subjected to oxygen–glucose deprivation (OGD). Lentiviral HIF-1α knockdown was assessed by reverse transcription–quantitative PCR (RT-qPCR) and western blotting. Cell Counting Kit-8 (CCK-8) signals, reactive oxygen species (ROS), and malondialdehyde (MDA) were measured. Protein interaction and enrichment analyses were combined with regulatory-network prediction and dual-luciferase reporter assays. HIF1A expression was elevated in the analyzed stroke-related datasets, and OGD increased HIF-1α expression in hCMEC/D3 cells. HIF-1α knockdown increased CCK-8 signals under OGD; however, MDA levels were higher in the shHIF-1α + OGD group than in the Control + OGD group. OGD increased ROS levels in both control and HIF-1α-knockdown cells. Reporter assays supported sequence-dependent interactions of miR-27a-3p with HIF1A-AS2 and HIF1A reporter constructs. These findings indicate that increased CCK-8 signals following HIF-1α knockdown coexist with increased MDA under OGD, rather than demonstrating uniform protection against oxidative injury. The reporter results suggest a potential regulatory relationship, but its contribution to the cellular phenotype requires functional rescue experiments. The divergent endpoints also warrant independent assessment of cell survival and lipid peroxidation before a protective role is assigned to HIF-1α knockdown.
Source: Research Square
Authors: Min Wang, Yabin Shen, Huaqiu Chen, Guangming Wang
Institutions: Dali University, Nantong University, Xichang University
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7225791069