Su Qiuxia, Dong Xianhua, Tang Chonghui, Wei Xiaojie, Hao Youguo, Wu Jun
University Healthcare Branch II, The First Affliated Hospital of Xiamen University, Xiamen, China.
Department of Neurosurgery, The First People's Hospital of Jiangxia District, Xiehe, Wuhan, Hubei, China.
Open Med (Wars). 2021 Apr 7;16(1):558-569. doi: 10.1515/med-2021-0209. eCollection 2021.
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 . 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.
动脉粥样硬化（AS）是一种严重的心血管疾病。环状RNA（circRNAs）在包括AS在内的许多疾病进展中发挥重要作用。然而，circ_0003204在AS中的作用尚不清楚。使用氧化型低密度脂蛋白（ox-LDL）诱导的人脐静脉内皮细胞（HUVECs）构建AS细胞模型。使用细胞计数试剂盒8（CCK8）检测法评估细胞活力。采用流式细胞术和半胱天冬酶-3活性检测细胞凋亡。使用酶联免疫吸附测定（ELISA）检测炎性细胞因子的含量。通过相应的检测试剂盒检测氧化应激标志物表达和细胞损伤标志物活性。此外，通过实时聚合酶链反应（qPCR）检测circ_0003204、miR-330-5p和Toll样受体4（TLR4）的表达水平。通过双荧光素酶报告基因检测和RNA下拉检测法检测miR-330-5p与circ_0003204或TLR4之间的相互作用。采用蛋白质免疫印迹（WB）分析确定TLR4蛋白和核因子-κB（NF-κB）信号通路相关蛋白的水平。我们的数据表明，ox-LDL可抑制HUVECs的活力并促进其凋亡、炎症反应和氧化应激。circ_0003204在ox-LDL诱导的HUVECs中高表达，其沉默可抑制ox-LDL诱导的HUVECs损伤。miR-330-5p可被circ_0003204吸附，其抑制剂可逆转沉默circ_0003204对ox-LDL诱导的HUVECs损伤的抑制作用。此外，TLR4可被miR-330-5p靶向，其过表达可逆转miR-330-5p对ox-LDL诱导的HUVECs损伤的抑制作用。NF-κB信号通路的活性受circ_0003204/miR-330-5p/TLR4轴调控。我们的结果表明，circ_0003204沉默可减轻ox-LDL诱导的HUVECs损伤，提示circ_0003204可能是AS治疗的新靶点。
