长链非编码RNA RP11-490M8.1对脂多糖诱导的人脐静脉内皮细胞炎症的影响

Long non-coding RNA RP11-490M8.1 inhibits lipopolysaccharide-induced inflammation in human vascular endothelial cells

目的 探讨长链非编码RNA(LncRNA)RP11-490M8.1在内皮细胞中对炎症因子的调控作用及其对动脉粥样硬化发生、发展的影响。方法 收集3对AS斑块组织和动脉内膜正常组织进行基因芯片分析,挖掘表达显著异常的基因;用脂多糖(lipopolysaccharide, LPS)浓度梯度和时间梯度处理内皮细胞、构建慢病毒载体过表达LncRNA RP11-490M8.1,以实时荧光定量PCR(qRT-PCR)检测LncRNA RP11-490M8.1和细胞炎症因子如肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、核转录因子-κB(NF-κB)的RNA水平,以免疫印迹(western blot)法检测细胞炎症因子(IL-6、TNF-α、NF-κB)的蛋白表达水平。最后将内皮细胞接种于6孔细胞培养板中,分为4组进行干预:(1)慢病毒空载体对照组(对照组):慢病毒空载体(LV-MOCK)转染内皮细胞培养24 h;(2)慢病毒空载体加LPS(LV-MOCK+LPS):LV-MOCK转染内皮细胞培养6 h,加1 000 ng/mL的LPS培养18 h;(3)慢病毒过表达组(LV-LncRNA RP11-490M8.1):LV-LncRNA RP11-490M8.1转染内皮细胞培养24 h;(4)LncRNA RP11-490M8.1过表达加LPS(LV-LncRNA RP11-490M8.1+LPS):LV-LncRNA RP11-490M8.1转染内皮细胞培养6 h,加1 000 ng/mL的LPS培养18 h。结果 基因芯片分析发现LncRNA RP11-490M8.1在AS斑块中表达下调,qRT-PCR和western blot结果显示LPS上调炎症因子(IL-6、TNF-α、NF-κB)的表达;慢病毒过表达LncRNA RP11-490M8.1后炎症因子表达下调;分组干预实验结果显示LncRNA RP11-490M8.1能减弱LPS对炎症因子的诱导作用。结论 在内皮细胞中LncRNA RP11-490M8.1抑制LPS诱导的炎症因子(IL-6、TNF-α、NF-κB)表达,LncRNA RP11-490M8.1很可能是药物发挥抗炎作用的靶向分子,为动脉粥样硬化的发展提供新的方向。

Objective To investigate the regulatory effects of long non-coding RNA RP11-490 M8.1 on inflammatory factors in human endothelial cells. Methods Three paired atherosclerotic plaques and normal arterial intima tissues were collected for microarray analysis to find genes with significant abnormal expression. The endothelial cells were treated with different concentrations and duration of lipopolysaccharide(LPS). Construction of lentiviral vector was used for overexpression of LncRNA RP11-490 m8.1. The RNA expressions of lncRNA RP11-490 m8.1 and inflammatory cytokines(IL-6, TNF-α, NF-κB) were assessed by quantitative real-time PCR(qRT-PCR), and the protein levels of inflammatory cytokines(IL-6, TNF-α, NF-κB) were assessed by Western blot. Endothelial cells were divided into four groups, Lentivirus empty vector(control group), in which cells were transfected with lentivirus empty vector(LV-Mock);LV-Mock+LPS group, in which cells were transfected with LV-Mock and treated with 1 000 ng/mL LPS;LV-LncRNA RP11-490 M8.1 group, in which cells were transfected with LV-LncRNA RP11-490 M8.1;and LV-LncRNA RP11-490 M8.1+LPS group, in which cells were transfected with LV-LncRNA RP11-490 M8.1 and treated with 1 000 ng/mL LPS. Results Microarray analysis showed that lncRNA RP11-490 M8.1 was low expressed in atherosclerotic plaques. LPS inhibited expression of lncRNA RP11-490 M8.1, while promoted expression of inflammatory factors. The levels of inflammatory factors were up-regulated by overexpression of lncRNA RP11-490 M8.1, indicating that lncRNA RP11-490 M8.1 negatively regulated inflammatory factors. The promotion of LPS on inflammatory factors was offset by overexpression of LncRNA RP11-490 M8.1. Conclusion These results demonstrated that lncRNA RP11-490 M8.1 inhibits LPS-induced inflammation in human vascular endothelial cells, indicating that LncRNA RP11-490 M8.1 is likely to be a target molecule for LPS to play inflammatory roles, providing new directions for the development of atherosclerosis.