CircUSP36 knockdown alleviates oxidized low‑density lipoprotein‑induced cell injury and inflammatory responses in human umbilical vein endothelial cells via the miR‑20a‑5p/ROCK2 axis

Abstract

The dysfunctions of human umbilical vein endothelial cells (HUVEcs) are important features of atherosclerosis (AS). circular RNAs (circRNAs) are regulators of a wide range of human diseases, including AS. The present study aimed to investigate the role of circUSP36 in the ectopic phenotype of HUVEcs and to provide evidence of the involvement of circUSP36 in the pathogenesis of AS. AS cell models in vitro were established using HUVEcs exposed to oxidized low-density lipoprotein (ox-LdL). cell viability, cell cycle progression and apoptosis, and cell migration and invasion were assessed by cell counting kit-8 (ccK-8) assay, flow cytometric assay and Transwell assay, respectively. The expression levels or releases of pro-inflammatory factors were detected by western blot analysis or enzyme-linked immunosorbent assay (ELISA). The mRNA expression of circUSP36, miR-20a-5p and Rho-associated coiled-coil kinase 2 (ROcK2) was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPcR), and the protein level of ROcK2 was detected by western blot analysis. The targeted association between miR-20a-5p and circUSP36 or ROCK2 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The results revealed that circUSP36 was highly expressed in ox-LdL-exposed HUVEcs. circUSP36 knockdown attenuated ox-LdL-induced cell cycle arrest, cell apoptosis and inflammatory responses, and promoted cell migration and invasion which had been blocked by ox-LdL. miR-20a-5p was found to be a target of circUSP36, and miR-20a-5p inhibition reversed the effects of circUSP36 knockdown. Moreover, miR-20a-5p directly bound to ROcK2, and miR-20a-5p inhibition aggravated ox-LdL-induced injury by increasing the ROcK2 level. More importantly, circUSP36 targeted miR-20a-5p to regulate the expression of ROcK2. On the whole, the present study demonstrates that, circUSP36 regulates ox-LdL-induced HUVEc injury and inflammation by modulating ROcK2 via competitively targeting miR-20a-5p.

A circular RNA, circUSP36, accelerates endothelial cell dysfunction in atherosclerosis by adsorbing miR-637 to enhance WNT4 expression

“…Furthermore, the potential of circUSP36 has been explored in the regulation of endothelial cell dysfunction. It has been proven that circUSP36 aggravates endothelial injury caused by ox-LDL by restraining cell proliferation, migration, invasion, and angiogenesis, while promoting apoptosis and inflammation [44][45][46][47]. In concert with these previous findings, this study demonstrated that circUSP36 was highly expressed in ox-LDLtreated endothelial cells than in the control group and that enforced expression of circUSP36 suppressed cell proliferation and migration in ox-LDL-treated endothelial cells.…”

Mechanisms of Oxidized LDL-Mediated Endothelial Dysfunction and Its Consequences for the Development of Atherosclerosis

“…Via targeting the miR-330-5p and further upregulating the expression of TLR4, the NF-κB pathway would be activated to regulate the inflammation response, oxidase stress, and cell apoptosis ( 36 ). The levels of vascular cell adhesion molecule 1 (VCAM1) and roundabout guidance receptor 1(ROBO1) was also upregulated with the miR-98-5p and miR-20a-5p downregulated and sponged by circ-USP36 in oxLDL-induced injuries, which could both accelerate the endothelial cell injury ( 37 , 38 ). However, differently, through absorbing the miR-637 to enhance WNT4, the circ-USP36 had been reported to attenuate the EC proliferation and migration in the oxLDL-induced injury ( 39 ).…”

Emerging Roles of Extracellular Vesicle-Delivered Circular RNAs in Atherosclerosis

“…In addition, exosomes from platelets, vessel cells ( He et al, 2018 ; Bai et al, 2020b ), inflammatory adipocytes ( Wadey et al, 2019 ) and dendritic cells increased the level of inflammatory factors and recruited inflammatory cells and promoted their adhesion to the vessel wall, leading to a chronic inflammatory response process in AS. Multiple circRNAs (circ_0004104 ( Zhang et al, 2021a ), circular ANRIL ( Song et al, 2017 ), circTM7SF3 ( Wang and Bai, 2021 ), circUSP36 ( Miao et al, 2021 ) and so on) were upregulated in AS and demonstrated to induce vascular EC injury and oxidative stress and inflammation. Circ_GRN and Circ_CHFR were also upregulated in atherosclerotic serum and expedited the inflammation of human vascular muscle cells ( Zhuang et al, 2020 ; Li et al, 2021a ).…”