MicroRNA let-7e对LPS诱导的TNF-α表达的影响及机制的初步研究  

Effects of let-7e on the LPS-induced expression of TNF-α and the possible mechanism

作　　者：张倩倩[1] 张英可[1] 桂连[2] 黄俊琪[1] 

机构地区：[1]中山大学附属第一医院广东省器官捐献与移植免疫重点实验室,广州510080 [2]中山大学中山医学院免疫学研究所,广州510080

出　　处：《中华微生物学和免疫学杂志》2015年第12期884-889,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30872350,31370870）;广东省自然科学基金资助项目（S2012010009050,s2013020013000,S81510008901000017）;广东省科技计划项目（2010B050700008,2011B040300022,2013A020229003）;广州市科技计划项目（11C32060749,2011J4100084,200821-E221）;2010年中山大学高校基本科研业务费青年培育项目（t0ykpy31）;广东省器官捐献与移植免疫重点实验室建设项目（2013A061401007）

摘　　要：目的研究let-7e对LPS诱导的人原代单核细胞产生的TNF-α表达的影响及其机制。方法用全血分离人原代单核细胞，流式细胞术检测单核细胞表面标记CD14；let-7e mimics或mimics NC瞬转原代单核细胞24h、36h、48h，qRT-PCR和免疫荧光法检测其转染效率；采用1mg／L LPS刺激原代单核细胞1h、3h、6h、12h后，ELISA法检测细胞培养上清中TNF-α的浓度，筛选出LPS最佳刺激时间；qRT—PCR法及ELISA法评估let-7e对LPS诱导原代单核细胞产生TNF-α的影响；Western blot法检测瞬转1et-7e mimics后EZH2的表达水平以及瞬转siEZH2后NF-κB p65、ARFGAP1和Arfaptin 2的表达水平。结果CD14^＋细胞大于70％；免疫荧光结果显示miRNA模拟物可以转染原代单核细胞，转染率约为70％；let-7e mimics转染原代单核细胞24h，细胞内即可表达较高水平的let-7e；LPS刺激原代单核细胞12h即可产生较高水平的TNF-α。ELISA结果证实let-7e mimics显著抑制LPS诱导的TNF-α的产生，同时在mRNA水平也得到一致的结果；Western blot结果显示let-7e mimics组EZH2的水平明显低于let-7e mimics NC组，沉默EZH2后胞核中NF—κ Bp65显著下调，沉默EZH2后囊泡转运调节蛋白ARFGAP1和Arfaptin2的表达显著下降。结论在原代单核细胞中，let-7e显著抑制LPS诱导的TNF-α的表达；其机制可能是1et-7e靶向作用EZH2进而调节NF—κB的活性及囊泡转运相关蛋白ARFGAP1和Arfaptin2的表达水平。Objective To study the effects of a miRNA family member, 1et-7e, on the LPS-induced expression of TNF-α in primary monocytes and the possible mechanism. Methods Peripheral blood mononuclear cells （PBMCs） were isolated form human blood sample by using density gradient eentrifugation for further isolation of primary monocytes. Flow eytomctry analysis was used to measure the purity of isolated primary monoeytes. The efficiencies of transfection were evaluated by qRT-PCR and immunofluorescence assay after transfecting the primary monocytes with 1et-7e mimic or miRNA mimic negative control （NC） for 24 h, 36 h and 48 h. To screen out the optimal stimulation time, ELISA was performed to detect the concentrations of TNF-α in the supernatants of cell culture after stimulating the primary monocytes with 1 mg/L of LPS for 0 h, 1 h, 3 h, 6 h and 12 h, respectively. ELISA and qRT-PCR were used to measure the expression of TNF-α in the transfected cells with the interference of LPS. Western blot assay was used to detect the level of enhancer of zeste homolog 2 （EZH2） in 1et-7e mimic-transfected primary monocytes and the levels of NF-κB p65, ADP-ribosylation factor GTPase-activating protein 1 （ ARFGAP1 ） and Arfaptin2 in the siEZH2- transfected monocytes. Results More than 70% of the isolated cells were CD14^＋ cells. The miRNA mimics could transfect the primary monocytes effectively and the transfection rate was about 70%. High levels of 1et- 7e were detected in 1et-7e mimic-transfected primary monocytes 24 hours after the transfection. High levels of TNF-α were observed in the primary monocytes after stimulated with LPS for 12 h, which was considered as the optimal LPS stimulation time. Results of the ELISA indicated that 1et-7e mimic significantly inhibited the LPS-induced expression of TNF-α in primary monocytes at both mRNA and protein levels. Western blot assay showed that the levels of EZH2 in the 1et-7e mimic transfected primary monocytes were significantly lower than that in mimic NC transfected

关 键 词：原代单核细胞 let-7e TNF-Α LPS 

分 类 号：R446.6[医药卫生—诊断学]