摘要
目的:探讨下调miR-217表达对类风湿性关节炎(RA)患者外周血单个核细胞(PBMC)炎症反应的影响及作用机制。方法:分离培养RA患者PBMC细胞,脂多糖(LPS)诱导PBMC细胞24 h,ELISA法检测炎症因子TNF-α、IL-1β、IL-6和IL-10的表达,qRT-PCR检测miR-217和Sirt1 mRNA表达,Western blot检测Sirt1蛋白表达。分别转染anti-miR-217和pcDNA3.1-Sirt1至PBMC细胞,构建miR-217低表达、Sirt1过表达的PBMC细胞,ELISA法检测PBMC细胞分泌TNF-α、IL-1β、IL-6和IL-10的变化。双荧光素酶报告基因验证miR-217与Sirt1的关系。结果:LPS诱导PBMC细胞24 h后,TNF-α、IL-1β和IL-6水平升高,IL-10水平降低。miR-217在LPS诱导的PBMC细胞中表达上调,Sirt1 mRNA和蛋白表达下调。下调miR-217表达或上调Sirt1表达均可抑制PBMC细胞TNF-α、IL-1β和IL-6表达,促进IL-10表达。miR-217在PBMC细胞中负向调控Sirt1表达,上调miR-217表达抑制了Sirt1过表达对LPS诱导的PBMC细胞中炎症因子表达的影响。结论:下调miR-217可能通过负向调控Sirt1表达抑制RA患者PBMC细胞炎症反应,可作为治疗RA的新靶点。
Objective:To investigate the effect of down-regulation of miR-217 expression on the inflammatory response of peripheral blood mononuclear cells(PBMC)in patients with rheumatoid arthritis(RA).Methods:PBMC cells from RA patients were isolated and cultured.Lipopolysaccharide(LPS)induced PBMC cells for 24 hours.The expression levels of inflammatory factors TNF-α,IL-1β,IL-6 and IL-10 were detected by ELISA.The levels of miR-217 and Sirt1 mRNA were detected by qRT-PCR.The level of Sirt1 protein was detected by Western blot.Anti-miR-217 and pcDNA3.1-Sirt1 were transfected to PBMC cells in order to construct PBMC cells with low expression of miR-217 and overexpression of Sirt1,ELISA was used to detect the levels of TNF-α,IL-1β,IL-6 and IL-10.The dual luciferase reporter gene verified the relationship between miR-217 and Sirt1.Results:After 24 hours of LPS-induced PBMC cells,TNF-α,IL-1βand IL-6 levels were increased,and IL-10 levels was decreased.miR-217 was up-regulated in LPS-induced PBMC cells,and Sirt1 mRNA and protein expression was down-regulated.Down-regulation of miR-217 expression or up-regulation of Sirt1 expression could inhibit the expression of TNF-α,IL-1βand IL-6 and promote IL-10 expression.miR-217 negatively regulated Sirt1 expression in PBMC cells,and up-regulation of miR-217 expression inhibited the effect of Sirt1 overexpression on LPS-induced inflammatory factor expression in PBMC cells.Conclusion:Down-regulation of miR-217 could inhibit the inflammatory response of PBMC cells in RA patients by negatively regulating Sirt1 expression,which could be a new target for the treatment of RA.
作者
郭淑芳
姜婷婷
GUO Shu-Fang;JIANG Ting-Ting(Medical School of Yan′an University,Yan′an 716000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第8期933-938,共6页
Chinese Journal of Immunology
