Abstract
Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3' untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.
Highlights
Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes
MiRNAs expressed in human aortic endothelial cell (HAEC) We sequenced four small RNA libraries prepared using total RNA from HAECs from two separate donors treated with media alone or media containing 40 g/ml oxidized 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphatidylcholine (Ox-PAPC) for 4 h
We detected between 386 and 583 miRNAs expressed per library, representing a total of 618 unique miRNAs expressed in HAECs
Summary
Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1 (IL-1). Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor B (NF-B) signaling Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. In vitro treatment of human aortic endothelial cells (HAECs) with a component of oxLDL, oxidized 1-palmitoyl-2-arachidonoyl-snglycero-3-phosphatidylcholine (Ox-PAPC), changes the expression of hundreds of genes involved in inflammation, unfolded protein response, coagulation, and sterol biosynthesis [3,4,5,6,7,8]. We hypothesized that miRNAs partially regulate the expression of genes that are involved in the response of ECs to Ox-PAPC.
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