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Circ_USP36 Silencing Attenuates Oxidized Low-Density Lipoprotein-Induced Dysfunction in Endothelial Cells in

Yixin Zhang¹, Wenhua Li², Hui Li¹

¹Department of Endocrinology, the Second Affiliated Hospital of Qiqihar Medical University, Qiqihar City, Heilongjiang, China; and.

Journal of Cardiovascular Pharmacology

|August 9, 2021

Insights

Circular RNA USP36 (circ_USP36) exacerbates endothelial cell dysfunction in atherosclerosis. Inhibiting circ_USP36 protects against oxidized LDL effects by targeting the miR-197-3p/ROBO1 pathway, offering a potential therapeutic strategy.

Area of Science:

Molecular Biology
Cardiovascular Research
Cell Biology

Background:

Circular RNAs (circRNAs) are increasingly recognized for their regulatory roles in cardiovascular diseases, including atherosclerosis.
Oxidized low-density lipoprotein (ox-LDL) is a key factor in endothelial cell (EC) dysfunction, a critical process in atherosclerosis development.
The specific role of circRNA ubiquitin-specific peptidase 36 (circ_USP36) in ox-LDL-induced EC dysfunction remains to be fully elucidated.

Purpose of the Study:

To investigate the biological role of circ_USP36 in oxidized low-density lipoprotein (ox-LDL)-induced dysfunction of human umbilical vein endothelial cells (HUVECs).
To elucidate the molecular mechanism underlying circ_USP36's function in this context, including its interaction with microRNA-197-3p (miR-197-3p) and roundabout guidance receptor 1 (ROBO1).

Main Methods:

Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot assays were used to measure RNA and protein levels.
Cell proliferation, cell cycle, apoptosis, and cell death were assessed using assays such as EdU, MTT, flow cytometry, and LDH.
Dual-luciferase reporter assays verified intermolecular interactions between circ_USP36, miR-197-3p, and ROBO1.

Main Results:

Circ_USP36 expression was significantly upregulated in atherosclerosis patients' serum and ox-LDL-stimulated HUVECs.
Knockdown of circ_USP36 ameliorated ox-LDL-induced inhibition of proliferation, cell cycle arrest, apoptosis, and inflammation in HUVECs.
Circ_USP36 targeted miR-197-3p, which in turn targeted ROBO1; circ_USP36 knockdown reduced ROBO1 expression by upregulating miR-197-3p.

Conclusions:

Circ_USP36 plays a detrimental role in ox-LDL-induced endothelial cell dysfunction.
Interference with circ_USP36 alleviates ox-LDL-induced HUVEC dysfunction by targeting the miR-197-3p/ROBO1 axis.
Circ_USP36 represents a potential therapeutic target for atherosclerosis.

Read full analysis

Abstract:

Circular RNAs (circRNAs) are reported to play pivotal regulatory roles in atherosclerosis progression. In the present study, we explored the biological role of circRNA ubiquitin-specific peptidase 36 (circ_USP36; hsa_circ_0003204) in oxidized low-density lipoprotein (ox-LDL)-induced dysfunction of endothelial cells (ECs). RNA and protein levels were determined by reverse transcription-quantitative polymerase chain reaction and Western blot assay, respectively. Cell proliferation was analyzed by 5-ethynyl-2'-deoxyuridine assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry was conducted to analyze cell cycle progression and cell apoptosis. The release of tumor necrosis factor α in the supernatant was measured by enzyme linked immunosorbent assay. Cell death was evaluated by lactate dehydrogenase assay. Intermolecular interaction was verified by dual-luciferase reporter assay. Circ_USP36 expression was significantly up-regulated in the serum of atherosclerosis patients and ox-LDL-stimulated HUVECs than that in their corresponding controls. ox-LDL exposure inhibited the proliferation ability and cell cycle progression and triggered the apoptosis and inflammation of HUVECs, and these effects were largely overturned by the knockdown of circ_USP36. microRNA-197-3p (miR-197-3p) was a target of circ_USP36, and circ_USP36 knockdown-mediated protective role in ox-LDL-induced HUVECs was largely counteracted by the silence of miR-197-3p. miR-197-3p interacted with the 3' untranslated region of roundabout guidance receptor 1 (ROBO1). Circ_USP36 knockdown reduced ROBO1 expression partly by up-regulating miR-197-3p in HUVECs. ROBO1 overexpression reversed miR-197-3p accumulation-mediated effects in ox-LDL-induced HUVECs. In conclusion, circ_USP36 interference alleviated ox-LDL-induced dysfunction in HUVECs by targeting miR-197-3p/ROBO1 axis.

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