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Updated: Aug 13, 2025
Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein
Published on: October 12, 2017
Circ_0033596 depletion ameliorates oxidized low-density lipoprotein-induced human umbilical vein endothelial cell
Yanling Teng1, Fei Ren1, Yanan Wang1
1Department of Cardiac Function, The First People's Hospital of Lianyungang, The First Affiliated Hospital of Kangda College of Nanjing Medical University, Lianyungang City, Jiangsu, China.
Insights
Circular RNA circ_0033596 promotes atherosclerosis by targeting the miR-637/GRB2 pathway. Depleting circ_0033596 protects against oxidized low-density lipoprotein-induced endothelial cell injury, offering a potential therapeutic strategy for atherosclerosis.
Area of Science:
- Cardiovascular Biology
- Molecular Medicine
- Cellular Pathology
Background:
- Atherosclerosis (AS) pathogenesis involves circ_0033596.
- The precise mechanism of circ_0033596 in AS requires elucidation.
Purpose of the Study:
- To investigate the detailed mechanism of circ_0033596 in AS.
- To explore the role of circ_0033596 in oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cell (HUVEC) injury.
Main Methods:
- Established an AS cell model using ox-LDL-treated HUVECs.
- Quantified gene and protein expression (circ_0033596, miR-637, GRB2, Bax, Bcl-2) via qPCR and Western blot.
- Assessed HUVEC viability, proliferation, apoptosis, and tube formation.
- Measured inflammatory cytokines (IL-6, TNF-α) and oxidative stress markers (MDA, SOD).
- Utilized dual-luciferase reporter, RNA pull-down, and RIP assays to confirm molecular interactions.
Main Results:
- circ_0033596 and GRB2 were upregulated, while miR-637 was downregulated in AS patients and ox-LDL-treated HUVECs.
- circ_0033596 knockdown attenuated ox-LDL-induced HUVEC damage, including apoptosis, inflammation, and oxidative stress.
- circ_0033596 directly targets miR-637, and GRB2 is a downstream target of miR-637.
- circ_0033596 promotes HUVEC injury by activating GRB2 via sponging miR-637.
Conclusions:
- circ_0033596 depletion protects HUVECs from ox-LDL-induced injury through the miR-637/GRB2 pathway.
- circ_0033596 represents a potential therapeutic target for atherosclerosis.
Background:
Previous data have shown that circ_0033596 is involved in the pathogenesis of atherosclerosis (AS). The study aims to reveal the detailed mechanism of circ_0033596 in AS.
Methods:
Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to establish an AS cell model. Quantitative real-time polymerase chain reaction and western blot were implemented to detect the expression of circ_0033596, miR-637, growth factor receptor bound protein2 (GRB2), BCL2-associated x protein (Bax) and B-cell lymphoma-2 (Bcl-2). Cell viability, proliferation, apoptosis and tube formation were investigated by cell counting kit-8, EdU assay, flow cytometry and tube formation assay, respectively. The production of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) was evaluated by enzyme-linked immunosorbent assay. Oxidative stress was evaluated by lipid peroxidation malondialdehyde assay kit and superoxide dismutase activity assay kit. Dual-luciferase reporter assay, RNA pull-down assay and RIP assay were performed to identify the associations among circ_0033596, miR-637 and GRB2.
Results:
The expression of circ_0033596 and GRB2 was significantly increased, while miR-637 was decreased in the blood of AS patients and ox-LDL-induced HUVECs compared with controls. Ox-LDL treatment inhibited HUVEC viability, proliferation and angiogenic ability and induced cell apoptosis, inflammation and oxidative stress, while these effects were attenuated after circ_0033596 knockdown. Circ_0033596 interacted with miR-637 and regulated ox-LDL-induced HUVEC damage by targeting miR-637. In addition, GRB2, a target gene of miR-637, participated in ox-LDL-induced HUVEC injury by combining with miR-637. Importantly, circ_0033596 activated GRB2 by interacting with miR-637.
Conclusion:
Circ_0033596 depletion protected against ox-LDL-induced HUVEC injury by miR-637/GRB2 pathway, providing a therapeutic target for AS.
