过表达miR-498通过下调STAT3抑制类风湿性关节炎患者Th17细胞分化
项海燕¹, 潘峰^2,*, 鄢巨振³, 洪理泉², 张腊红², 刘玉华², 冯晓², 蔡成松^2
1杭州师范大学附属医院静配中心，杭州 310015；²杭州师范大学附属医院医学检验科，杭州 310015；³杭州师范大学附属医院肾病风湿免疫科，杭州 310015
摘要
本文旨在探讨微小RNA-498 (miR-498)影响类风湿性关节炎(rheumatoid arthritis, RA)患者外周血单核细胞(peripheral blood mononuclear cells, PBMCs)中Th17细胞分化的作用机制。从50例RA患者和50例对照人群中分别收集外周血，分离PBMCs。流式细胞术检测CD4+IL-17+ T (Th17)细胞或者CD4+ FOXP3+ T (Treg)细胞比例，并分析Th17/Treg比值。Real-time PCR检测细胞中miR-498和STAT3基因表达，Western blot检测STAT3的蛋白表达，ELISA检测外周血血清或细胞上清中IL-17的浓度。荧光素酶报告基因实验验证miR-498靶向结合STAT3的3’非翻译(3’untranslated region, 3’UTR)。miR-498 mimic或/和pcDNA-STAT3瞬时转染CD4+ T细胞，进一步考察miR-498影响Th17细胞分化的机制。结果显示，RA患者的PBMCs中，Th17细胞比例显著高于对照，且Th17/Treg比值也显著高于对照(P < 0.05)。miR-498低表达而STAT3高表达于RA患者的PBMCs中，且RA患者血清中的IL-17浓度显著高于对照(P < 0.05)。在转染野生型STAT3报告基因质粒的细胞中转染miR-498 mimic/inhibitor后，荧光酶活性、STAT3的基因和蛋白表达均明显改变(P < 0.05)，但是在转染突变型STAT3报告基因质粒的细胞中转染miR-498 mimic/inhibitor，上述指标没有显著变化(P > 0.05)。此外，miR-498 mimic转染CD4+ T细胞后，显著降低Th17细胞比例，且细胞分泌IL-17的水平减少(P < 0.05)，而与pcDNA-STAT3共转CD4+ T细胞后，Th17细胞比例明显提高，细胞分泌IL-17的水平增加(P < 0.05)。以上结果提示，在RA患者CD4+ T细胞中，过表达miR-498靶向下调STAT3，进而抑制Th17细胞分化。
关键词： 微小RNA-498; STAT3; Th17 细胞分化; 类风湿性关节炎
分类号：R392；R593

Upregulation of miR-498 suppresses Th17 cell differentiation by targeting STAT3 in rheumatoid arthritis patients
XIANG Hai-Yan¹, PAN Feng^2,*, YAN Ju-Zhen³, HONG Li-Quan², ZHANG La-Hong², LIU Yu-Hua², FENG Xiao², CAI Cheng-Song^2
1Pharmacy Intravenous Admixture Service, The Affiliated Hospital of Hangzhou Normal University, Hangzhou 310015, China；²Department of Clinical Laboratory, The Affiliated Hospital of Hangzhou Normal University, Hangzhou 310015, China；³Department of Nephrology and Rheumatology, The Affiliated Hospital of Hangzhou Normal University, Hangzhou 310015, China
Abstract
To investigate the effect and mechanism of miR-498 on Th17 cell differentiation of peripheral blood mononuclear cells (PMBCs) in rheumatoid arthritis (RA) patients, peripheral blood samples were collected from RA patients and healthy controls, respectively. The proportion of CD4+IL-17+ T cells (Th17 cells) or CD4+FOXP3+ T cells (Tregs) in T cells and the Th17/Treg ratio were identified by the flow cytometer. The STAT3 and miR-498 expression were measured by Western blot and real-time PCR, respectively. ELISA was used to detect IL-17 concentrations. Luciferase assay was performed to confirm that miR-498 directly targeted the 3’ untranslated region (3’UTR) of STAT3 in CD4+ T cells. The effect of miR-498 on Th17 cell differentiation was explored by transfection of miR-498 mimic and/or pcDNA-STAT3 into CD4+ T cells. In PMBCs of RA patients, the Th17/CD4+ T cell ratio was significantly increased, while the Tregs/CD4+ T cell ratio was obviously decreased, leading to a higher Th17/Treg ratio. The results showed a reduced miR-498 expression and an increased STAT3 protein expression in PMBCs, and an increased IL-17 concentration in serum of RA patients. In cells transfected with wild-type-STAT3-LU, miR-498 mimic significantly reduced the luciferase activity, STAT3 gene and protein expression, and miR-498 inhibitor had an opposite function. While the miR-498 mimic/inhibitor had no effect on the luciferase activity and STAT3 expression in cells transfected with mutant-STAT3-LU. CD4+ T cells transfected with miR-498 mimic had a lower Th17/CD4+ T cell ratio and IL-17 concentration, however, transfection of pcDNA-STAT3 reversed the effect of miR-498 mimic on Th17/CD4+ T cell ratio and IL-17 concentration. These results suggest that overexpression of miR-498 suppresses Th17 cell differentiation by targeting STAT3 in RA patients.
Key words: miR-498; STAT3; Th17 cell differentiation; rheumatoid arthritis

收稿日期：2017-07-20　　录用日期：2018-02-05
通讯作者：潘峰　　E-mail: Panfeng135@163.com
DOI: 10.13294/j.aps.2018.0019
引用本文：
项海燕, 潘峰, 鄢巨振, 洪理泉, 张腊红, 刘玉华, 冯晓, 蔡成松. 过表达miR-498通过下调STAT3抑制类风湿性关节炎患者Th17细胞分化[J]. 生理学报 2018; 70 (2): 167-174.
XIANG Hai-Yan, PAN Feng, YAN Ju-Zhen, HONG Li-Quan, ZHANG La-Hong, LIU Yu-Hua, FENG Xiao, CAI Cheng-Song. Upregulation of miR-498 suppresses Th17 cell differentiation by targeting STAT3 in rheumatoid arthritis patients. Acta Physiol Sin 2018; 70 (2): 167-174 (in Chinese with English abstract).



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