基于Jurkat细胞分析circ_0006689对系统性红斑狼疮中IL2的作用及机制研究

Effect and mechanism of circ_0006689 on IL2 in systemic lupus erythematosus based on Jurkat cells

  • 摘要: 目的:在Jurkat细胞基础上初步探讨circ_0006689在系统性红斑狼疮(SLE)中调控白介素2(IL2)的作用机制。方法:使用慢病毒转染Jurkat 细胞构建circ_0006689 低表达株。用PMA、PHA 活化低表达组(si-circ_0006689 组)、病毒空载组(si-NC组)及未转染组(control组)细胞后通过实时荧光定量PCR(RT-qPCR)检测各组IL2、miR-95-5p的相对表达量;酶联免疫吸附试验(ELISA)法检测各组细胞IL2浓度;生物信息学分析预测circ_0006689的目标miRNA及IL2的目标miRNA;双荧光素酶报告实验检测circ_0006689与miR-95-5p、miR-95-5p与IL2的靶向关系。结果:Jurkat细胞活化后IL2分泌显著增加(P< 0.05);下调circ_0006689 后细胞IL2 表达及分泌减少(P< 0.05),miR-95-5p 表达水平显著升高(P< 0.05)。双荧光素酶报告实验提示circ_0006689 与miR-95-5p、miR-95-5p 与IL2 存在结合作用。结论:在Jurkat 细胞中低表达circ_0006689 可抑制IL2 的表达;miR-95-5p可分别与circ_0006689、IL2结合;circ_0006689可能在SLE中通过竞争性结合miR-95-5p影响IL2的表达及分泌。

     

    Abstract: Objective:To preliminarily explore the effect and mechanism of circ_0006689 in regulating interleukin 2(IL2)in systemic lupus erythematosus(SLE)on the basis of Jurkat cells.Methods:Jurkat cells were transfected with lentivirus to construct circ_0006689 low expression strain; the relative expression levels of IL2 and miR-95-5p were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) after PMA and PHA activated the cells in low expression group (si-circ_0006689 group), virus-free group (si-NC group)and untransfected group(control group).The concentration of secreted IL2 in each group was detected by enzyme-linked immunosorbent assay (ELISA); bioinformatics analysis predicted the target miRNA of circ_0006689 and the target miRNA of IL2; dual-luciferase reporter assay was used to detect the targeting relationship between circ_0006689 and miR-95-5p, and miR-95-5p and IL2.Results:IL2 secretion significantly increased after Jurkat cell activation (P< 0.05); circ_0006689 was down-regulated, IL2 expression and secretion decreased(P< 0.05), and the expression level of miR-95-5p increased significantly (P< 0.05).The dual-luciferase reporter assay suggested the binding effect of circ_0006689 to miR-95-5p and miR-95-5p to IL2.Conclusion:Low expression of circ_0006689 in Jurkat cells can inhibit IL2 expression; miR-95-5p can bind to circ_0006689 and IL2, respectively; the circ_0006689 may affect IL2 expression and secretion by competitive binding to miR-95-5p in SLE.

     

/

返回文章
返回