Knockdown of Circular Ubiquitin-specific Peptidase 9 X-Linked Alleviates Oxidized Low-density Lipoprotein-induced Injuries of Human Umbilical Vein Endothelial Cells by Mediating the miR-148b-3p/KLF5 Signaling Pathway

Abstract: There is evidence that the development of atherosclerosis (AS) involves the dysregulation of circular RNAs. This study aimed to investigate the role of circular ubiquitin-specific peptidase 9 X-linked (circUSP9X) in AS cell models. Human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (ox-LDL) were used as cell models of AS. The expression of circUSP9X, miR-148b-3p, and Kruppel-like factor 5 (KLF5) messenger RNA was measured using quantitative polymerase chain reaction. Cell viability was assessed by Cell Counting Kit-8 assay. Lactate dehydrogenase leakage, malonaldehyde content, and superoxide dismutase activity were investigated using matched commercial kits. Cell apoptosis was detected using flow cytometry assay. The protein levels of apoptosis-related markers and KLF5 were detected by western blot. The release of proinflammatory factors was monitored by enzyme-linked immunosorbent assay. The predicted relationship between miR-148b-3p and circUSP9X or KLF5 was confirmed by dual-luciferase reporter assay or RNA immunoprecipitation assay. CircUSP9X was highly expressed in ox-LDL–treated HUVECs. CircUSP9X knockdown inhibited ox-LDL–induced lactate dehydrogenase leakage, apoptosis, inflammation, and oxidative stress in HUVECs. CircUSP9X directly bound to miR-148b-3p, and KLF5 was a target of miR-148b-3p. CircUSP9X could regulate KLF5 expression by competitively targeting miR-148b-3p. Rescue experiments indicated that circUSP9X knockdown inhibited ox-LDL–induced HUVEC injuries by enriching miR-148b-3p, and miR-148b-3p restoration alleviated ox-LDL–induced HUVEC injuries by degrading KLF5. In conclusion, circUSP9X knockdown relieved ox-LDL–triggered HUVEC injuries during AS progression partly by mediating the miR-148b-3p/KLF5 network.

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