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Updated: Jul 7, 2025
Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
Methylation and transcriptomic expression profiles of HUVEC in the oxygen and glucose deprivation model and its
Yuning Tang1,2,3,4, Yongxiang Wang2,4,5, Shengxiang Wang6
1The First School of Clinical Medicine, Lanzhou University, Lanzhou, China.
Insights
This study reveals how DNA methylation changes in endothelial cells during ischemia-reperfusion injury impact gene expression, identifying potential biomarkers for acute myocardial infarction (AMI). These findings link cellular changes to clinical outcomes in AMI patients.
Area of Science:
- Molecular Biology
- Genomics
- Cardiovascular Research
Background:
- Acute myocardial infarction (AMI) involves pathological changes in coronary arteries due to ischemic-hypoxic shocks.
- The role of DNA methylation in endothelial cells during AMI and its impact on disease pathology remain underexplored.
Purpose of the Study:
- To investigate the relationship between DNA methylation and altered gene expression in human umbilical vein endothelial cells (HUVECs) under oxygen-glucose deprivation (OGD).
- To explore the clinical implications of these findings in patients with AMI.
Main Methods:
- Genome-wide DNA methylation profiling using Illumina Infinium MethylationEPIC BeadChip and mRNA sequencing.
- Bioinformatic analyses including Gene Ontology (GO), KEGG pathway enrichment, correlation, causal inference test (CIT), and protein-protein interaction (PPI).
- Validation using MethylTarget sequencing, qRT-PCR, and ELISA for target molecule detection in cell cultures and patient blood samples.
Main Results:
- Identified 2,524 differentially expressed genes (DEGs) and 22,148 differentially methylated positions (DMPs) corresponding to 6,642 differentially methylated genes (DMGs).
- Filtered 441 genes through comprehensive bioinformatic analyses, identifying 22 hub genes.
- Confirmed elevated levels of TNFSF10 and BDNF in the peripheral blood of AMI patients, with BDNF expression shown to be regulated by DNA methylation in endothelial cells during ischemia/hypoxia.
Conclusions:
- Established a potential causal chain from DNA methylation to mRNA expression and endothelial cell injury, linking to clinical outcomes in AMI.
- Identified OGD-specific genes as potential diagnostic and prognostic biomarkers for coronary endothelial cell injury in AMI.
- Provided the first evidence of DNA methylation regulating BDNF expression in endothelial cells under ischemic conditions, with elevated BDNF in AMI patients.
Abstract:
The obstructed coronary artery undergoes a series of pathological changes due to ischemic-hypoxic shocks during acute myocardial infarction (AMI). However, the altered DNA methylation levels in endothelial cells under these conditions and their implication for the etiopathology of AMI have not been investigated in detail. This study aimed to explore the relationship between DNA methylation and pathologically altered gene expression profile in human umbilical vein endothelial cells (HUVECs) subjected to oxygen-glucose deprivation (OGD), and its clinical implications in AMI patients. The Illumina Infinium MethylationEPIC BeadChip assay was used to explore the genome-wide DNA methylation profile using the Novaseq6000 platform for mRNA sequencing in 3 pairs of HUVEC-OGD and control samples. GO and KEGG pathway enrichment analyses, as well as correlation, causal inference test (CIT), and protein-protein interaction (PPI) analyses identified 22 hub genes that were validated by MethylTarget sequencing as well as qRT-PCR. ELISA was used to detect four target molecules associated with the progression of AMI. A total of 2,524 differentially expressed genes (DEGs) and 22,148 differentially methylated positions (DMPs) corresponding to 6,642 differentially methylated genes (DMGs) were screened (|Δβ|>0.1 and detection p < 0.05). After GO, KEGG, correlation, CIT, and PPI analyses, 441 genes were filtered. qRT-PCR confirmed the overexpression of VEGFA, CCL2, TSP-1, SQSTM1, BCL2L11, and TIMP3 genes, and downregulation of MYC, CD44, BDNF, GNAQ, RUNX1, ETS1, NGFR, MME, SEMA6A, GNAI1, IFIT1, and MEIS1. DNA fragments BDNF_1_ (r = 0.931, p < 0.0001) and SQSTM1_2_NEW (r = 0.758, p = 0.0043) were positively correlated with the expressions of corresponding genes, and MYC_1_ (r = -0.8245, p = 0.001) was negatively correlated. Furthermore, ELISA confirmed TNFSF10 and BDNF were elevated in the peripheral blood of AMI patients (p = 0.0284 and p = 0.0142, respectively). Combined sequencing from in vitro cellular assays with clinical samples, aiming to establish the potential causal chain of the causal factor (DNA methylation) - mediator (mRNA)-cell outcome (endothelial cell ischemic-hypoxic injury)-clinical outcome (AMI), our study identified promising OGD-specific genes, which provided a solid basis for screening fundamental diagnostic and prognostic biomarkers of coronary endothelial cell injury of AMI. Moreover, it furnished the first evidence that during ischemia and hypoxia, the expression of BNDF was regulated by DNA methylation in endothelial cells and elevated in peripheral blood.
