Abstract
Atherosclerosis (AS) is a serious cardiovascular disease. Circular RNAs (circRNAs) play an important role in the progression of many diseases, including AS. However, the role of circ_0003204 in AS is not clear. Oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) were used to construct an AS cell model in vitro. Cell viability was assessed using cell counting kit 8 (CCK8) assay. Flow cytometry and caspase-3 activity were used to measure cell apoptosis. The contents of inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA). Oxidative stress marker expression and cell injury marker activity were detected by their corresponding Assay Kits. Besides, the expression levels of circ_0003204, miR-330-5p, and toll-like receptor 4 (TLR4) were tested by real-time polymerase chain reaction (qPCR). The interaction between miR-330-5p and circ_0003204 or TLR4 was examined by dual-luciferase reporter assay and RNA pull-down assay. Western blot (WB) analysis was used to determine the levels of TLR4 protein and nuclear factor-kappa B (NF-κB) signaling pathway-related protein. Our data suggested that ox-LDL could suppress viability and promote apoptosis, inflammatory response, and oxidative stress in HUVECs. circ_0003204 was highly expressed in ox-LDL-induced HUVECs, and its silencing could inhibit ox-LDL-induced HUVECs injury. miR-330-5p could be sponged by circ_0003204, and its inhibitor could reverse the inhibition effect of silenced circ_0003204 on ox-LDL-induced HUVECs injury. Further, TLR4 could be targeted by miR-330-5p, and its overexpression could invert the suppression effect of miR-330-5p on ox-LDL-induced HUVECs injury. The activity of the NF-κB signaling pathway was regulated by the circ_0003204/miR-330-5p/TLR4 axis. Our results indicated that circ_0003204 silencing could alleviate ox-LDL-induced HUVECs injury, suggesting that circ_0003204 might be a novel target for AS treatment.
Highlights
Atherosclerosis (AS) is a harmful condition caused by the accumulation of fat, blood clots, connective tissue, and calcium carbonate in blood vessels [1,2]
Through measuring the expression of toll-like receptor 4 (TLR4), we discovered that Oxidized low-density lipoprotein (ox-LDL) could remarkably promote TLR4 expression in human umbilical vein endothelial cells (HUVECs) (Figure 5e)
With the deepening of research, researchers are exploring new targets for the treatment of AS. circRNA has received a lot of attention as a potential therapeutic target for many diseases, mainly because its expression is closely related to disease progression [25,26]
Summary
Atherosclerosis (AS) is a harmful condition caused by the accumulation of fat, blood clots, connective tissue, and calcium carbonate in blood vessels [1,2]. Oxidized low-density lipoprotein (ox-LDL) has been proved to be a vital risk factor for AS, and ox-LDL-induced human umbilical vein endothelial cells (HUVECs) apoptosis and injury have been used as a good way to construct an in vitro cell model of AS [8,9]. Elucidating the causes affecting ox-LDL-induced HUVECs apoptosis and injury is of great clinical significance to reveal the pathogenesis of AS. All plasmids and oligonucleotides were purchased from GenePharma (Shanghai, China) and transfected into HUVECs by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) They were listed as follows: circ_0003204 small interfering RNA and overexpression plasmid (si-circ_0003204 and circ_0003204) or their negative controls (si-NC and pcDNA), miR-330-5p mimic and inhibitor (miR-330-5p and in-miR330-5p) or their negative controls (miR-NC and in-miRNC), TLR4 overexpression plasmid (TLR4) and its negative control (pcDNA)
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