Chen S, Wang Y, Qin H, Lin J, Xie L, Chen S, Liang J, Xu J
1 Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, China.
2 Shanghai Institute of Dermatology, Shanghai, China.
Lupus. 2019 Apr;28(4):510-519. doi: 10.1177/0961203319829853. Epub 2019 Feb 13.
Accumulating evidence suggests that the AKT/mTOR pathway plays an important role in the pathogenesis of systemic lupus erythematosus (SLE) through activating T cells, and there are few studies looking into the role of microRNA (miRNAs) in the mechanism. We first found that miR-633 expression in CD4T cells of SLE patients was significantly reduced.
To investigate the role of miR-633 in the AKT/mTOR pathway in lupus CD4T cells.
Samples of 17 SLE cases and 16 healthy controls were collected to detect the expression of miR-633, AKT1, mTOR mRNA and proteins by quantitative polymerase chain reaction (qPCR) and Western-blot, respectively. To determine whether AKT1 is a direct target of miR-633, a luciferase assay was performed. In vitro, AKT1 siRNA, miR-633 mimics/inhibitors or negative controls were transfected to Jurkat cells, human primary CD4T cells and lupus CD4T cells. RNA and proteins were extracted after 48 h, and levels of AKT/mTOR pathway markers and downstream multiple cytokines were detected by qPCR or Western-blot.
In SLE patients, the miR-633 levels in CD4T cells were significantly decreased and negatively correlated with SLEDAI. AKT1, mTOR mRNA and proteins were all up-regulated. The degree of downregulation of miR-633 was correlated negatively with AKT1 mRNA. The luciferase assay proved that AKT1 is a direct target of miR-633. In Jurkat and lupus CD4T cells, overexpression of miR-633 could result in lower levels of AKT1 and mTOR. Inhibition of miR-633 expression in primary CD4T cells caused reverse effects, and protein levels of p-AKT, p-mTOR, and p-S6RP increased. Moreover, among various cytokines, the expression of IL-4, IL-17, and IFN-γ mRNA was raised.
Our study suggests that miR-633 deletion can activate the AKT/mTOR pathway by targeting AKT1 to participate in the pathogenesis of SLE.
越来越多的证据表明，AKT/mTOR信号通路通过激活T细胞在系统性红斑狼疮（SLE）的发病机制中起重要作用，而关于微小RNA（miRNA）在该机制中的作用研究较少。我们首次发现SLE患者CD4+T细胞中miR-633表达显著降低。
探讨miR-633在狼疮CD4+T细胞的AKT/mTOR信号通路中的作用。
收集17例SLE患者和16例健康对照的样本，分别采用定量聚合酶链反应（qPCR）和蛋白质免疫印迹法检测miR-633、AKT1、mTOR mRNA及蛋白表达。为确定AKT1是否为miR-633的直接靶点，进行荧光素酶报告基因实验。体外实验中，将AKT1 siRNA、miR-633模拟物/抑制剂或阴性对照转染至Jurkat细胞、人原代CD4+T细胞和狼疮CD4+T细胞。48小时后提取RNA和蛋白质，通过qPCR或蛋白质免疫印迹法检测AKT/mTOR信号通路标志物及下游多种细胞因子水平。
SLE患者CD4+T细胞中miR-633水平显著降低，且与SLE疾病活动指数（SLEDAI）呈负相关。AKT1、mTOR mRNA及蛋白均上调。miR-633下调程度与AKT1 mRNA呈负相关。荧光素酶报告基因实验证明AKT1是miR-633的直接靶点。在Jurkat细胞和狼疮CD4+T细胞中，miR-633过表达可导致AKT1和mTOR水平降低。抑制原代CD4+T细胞中miR-633表达产生相反作用，p-AKT、p-mTOR和p-S6RP蛋白水平升高。此外，在多种细胞因子中，IL-4、IL-17和IFN-γ mRNA表达升高。
我们的研究表明，miR-633缺失可通过靶向AKT1激活AKT/mTOR信号通路，参与SLE的发病机制。
