微小RNA-146a调控肺泡巨噬细胞中白细胞介素-1受体相关激酶1和肿瘤坏死因子受体相关因子6的表达  

The effect of transfected microRNA-146a on expression of interleukin-1 receptor-associated kinase 1 and tumor necrosis factor receptor-associated factor 6 in alveolar macrophages

作　　者：黄萍[1] 刘芬[2] 曾振国[2] 黄彩雪 邵强[2] 夏亮[2] 揭克敏[3] 钱克俭[2] 

机构地区：[1]南昌大学第一附属医院、国家药物临床试验机构,江西330006 [2]南昌大学第一附属医院重症医学科,江西330006 [3]南昌大学医学院生物化学与分子生物学教研室

出　　处：《中华危重病急救医学》2014年第5期300-303,共4页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81060152）;国家自然科学基金（81160233）;国家自然科学基金青年基金项目（81101410）

摘　　要：目的 观察转染微小RNR-146a（miR-146a）对肺泡巨噬细胞中白细胞介素-1受体相关激酶1（IRAK-1）和肿瘤坏死因子受体相关因子6（TRAF-6）表达的影响,为探讨miR-146a对肺泡巨噬细胞炎症反应的调控机制奠定基础.方法 将体外培养大鼠肺泡巨噬细胞NR8383分为miR-146a模拟物（mimic）组和阴性对照组,分别加入50 nmol/L Pre-miR miR-146a前体和Cy3标记的Pre-miR阴性对照进行转染,转染24 h后收集细胞,采用实时荧光定量逆转录-聚合酶链反应（RT-qPCR）检测细胞中miR-146a、IRAK-1 mRNA、TRAF-6mRNA的表达,采用蛋白质免疫印迹试验（Western Blot）检测细胞中IRAK-1和TRAF-6的蛋白表达.结果 miR-146amimic组miR-146a表达较阴性对照组上调了（24.55±6.14）倍（t=-6.643,P=0.003）.细胞中IRAK-1和TRAF-6的mRNA表达分别为阴性对照组的（1.16±0.10）倍（t=2.701,P=0.054）和（1.19±0.16）倍（t=2.032,P=0.112）.而IRAK-1和TRAF-6的蛋白表达分别为阴性对照组的73.0％（t=-9.353,P=0.001）和64.1％（t=-6.839,P=0.002）.结论 miR-146a mimic能成功转染肺泡巨噬细胞NR8383; miR-146a过表达后,能有效下调肺泡巨噬细胞中IRAK-1和TRAF-6的表达,其机制可能是在蛋白翻译水平的抑制.Objective To observe the effect of transfected microRNA-146a （miR-146a） on expression of interleukin-1 receptor-associated kinase 1 （IRAK-1） and tumor necrosis factor receptor-associated factor 6 （TRAF-6） in alveolar macrophages,and to explore the regulatory mechanism of miR-146a in the inflammatory response of alveolar macrophages.Methods Alveolar macrophages NR8383 were cultured and divided into two groups：transfected miR-146a mimic group was transfected 50 nmol/L Pre-miR miR-146a precursors and the negative control group was transfected Cy3-labeled Pre-miR negative control.Cells were collected at 24 hours after transfection.The miR-146a and the mRNA expression of IRAK-1 and TRAF-6 were detected by real-time fluorescent quantitation reverse transcription-polymerase chain reaction （RT-qPCR）,and the protein expression of IRAK-1 and TRAF-6 was assayed by Western Blot.Results Compared with negative control group,the expression of miR-146a was upregulated by （24.55 ± 6.14） fold compared with miR-146a mimic group （t=-9.353,P=0.001）.The mRNA expressions of IRAK-1 and TRAF-6 in miR-146a mimic group were upregulated by （1.16 ± 0.10） fold （t=2.701,P=0.054） and （1.19 ± 0.16） fold （t=2.032,P=0.112）,respectively,compared with that of negative control group,but the protein levels of IRAK-1 and TRAF-6 were decreased by 73.0％ （t =-9.353,P =0.001） and 64.1％ （t =-6.839,P =0.002）,respectively.Conclusions miR-146a mimic was successfully transfected into the alveolar macrophage NR8383.The overexpression of miR-146a in alveolar macrophages can down-regulate the expression of IRAK-1 and TRAF-6 in protein translation levels,and its mechanism may be related with inhibition of protein translation.

关 键 词：微小RNA-146a 白细胞介素-1受体相关激酶1 肿瘤坏死因子受体相关因子6 肺泡巨噬细胞 炎症反应 

分 类 号：R563[医药卫生—呼吸系统]