CircNMD3 relieves endothelial cell injury induced by oxidatively modified low-density lipoprotein through regulating miR-498/ BMP and activin membrane-bound inhibitor (BAMBI) axis

Atherosclerosis (AS) is one of the most common vascular diseases. The endothelial injury theory indicates that atherosclerotic plaque is the result of endothelial cell injury. Recent studies have revealed that circRNAs are abnormally expressed in AS cell models, which are implicated in the regulation of various cell behaviors. In this study, we showed the downregulation of circNMD3 in AS, and studied its role in the model of endothelial cell injury by proliferation and apoptosis assay, caspase 3 activity assay, and ELISA. We also identified and validated its downstream targets by luciferase reporter assay, RNA pull-down experiment, Western blot, and RT-qPCR. CircNMD3 overexpression or miR-498 knockdown could inhibit the ox-LDL (oxidatively modified low-density lipoprotein)-induced injury in HUVEC (human umbilical vein endothelial cells), while the co-transfection of miR-498 mimic or siRNA targeting BAMBI (BMP and activin membrane bound inhibitor) attenuated the protective effect of circNMD3 overexpression. Overall, our data suggest that circNMD3 regulates the miR-498/BAMBI axis in endothelial cells to protect ox-LDL-induced damages. As a molecular sponge of miR-498, circNMD3 regulates the level of miR-498, which in turn modulates BAMBI expression and suppresses ox-LDL-induced injury in HUVECs.

动脉粥样硬化（Atherosclerosis, AS）是最常见的血管疾病之一。内皮损伤学说指出，动脉粥样硬化斑块的形成源于内皮细胞损伤。近期研究证实，环状RNA（circRNAs）在AS细胞模型中存在异常表达，并参与调控多种细胞行为。本研究发现circNMD3在AS中呈下调表达，并通过增殖与凋亡检测、半胱天冬酶3活性检测及酶联免疫吸附测定（ELISA），探究其在内皮细胞损伤模型中的作用。本研究同时通过荧光素酶报告基因检测、RNA下拉实验、蛋白质印迹（Western blot）及实时定量聚合酶链式反应（RT-qPCR），鉴定并验证了circNMD3的下游靶标。实验结果显示，circNMD3过表达或微小RNA-498（miR-498）敲低，均可抑制氧化低密度脂蛋白（ox-LDL, oxidatively modified low-density lipoprotein）诱导的人脐静脉内皮细胞（HUVEC, human umbilical vein endothelial cells）损伤；但若共转染miR-498模拟物或靶向BMP与激活素膜结合抑制剂（BAMBI, BMP and activin membrane bound inhibitor）的小干扰RNA（siRNA），则会削弱circNMD3过表达所带来的保护作用。综上，本研究数据表明，circNMD3可通过在内皮细胞中调控miR-498/BAMBI信号轴，对抗ox-LDL诱导的细胞损伤。作为miR-498的分子海绵，circNMD3可调节miR-498的表达水平，进而调控BAMBI的表达，最终抑制ox-LDL诱导的HUVEC损伤。