lncRNA MIAT在巨噬细胞炎症反应中的作用  

Effects of lncRNA MIAT on macrophage inflammatory reaction

作　　者：王子叶 杨堃[2] 温大蔚 赵磊[1] 王吉波(指导)[1] WANG Zi-Ye;YANG Kun;WEN Da-Wei;ZHAO Lei;WANG Ji-Bo(Department of Rheumatology&Immunology,Qingdao University Affiliated Hospital,Qingdao 216000,China)

机构地区：[1]青岛大学附属医院风湿免疫科,青岛216000 [2]青岛大学附属医院医学研究中心,青岛216000

出　　处：《中国免疫学杂志》2020年第16期1921-1924,1930,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金(81671603,81201060);山东省自然科学基金(ZR2016HM56)资助。

摘　　要：目的:探索长链非编码RNA心肌梗死转录本(lncRNA MIAT)在巨噬细胞炎症反应中的作用及其可能参与的炎症通路。方法:脂多糖(LPS)刺激小鼠巨噬细胞J774A.1制备炎症反应细胞模型(LPS组),以等体积PBS刺激的J774A.1细胞为PBS对照组(PBS组),实时定量PCR(RT-qPCR)及ELISA法检测两组IL-1β、TNF-α的mRNA及蛋白表达水平,RT-qPCR检测lncRNA MIAT在两组的表达量及亚细胞定位;shRNA敲低lncRNA MIAT,分为shMIAT敲低(KD)组、shNC阴性对照(NC)组,RT-qPCR检测lncRNA MIAT敲低效率及两组IL-1β、TNF-αmRNA相对表达量;Western blot检测KD、NC组的NF-κB、ERK5等多种转录因子磷酸化水平;在LPS刺激J774A.1细胞分别加入ERK5特异性抑制剂(ERK5-IN-2)、ERK5-IN-2溶解液二甲基亚砜(DMSO),分为抑制剂组、DMSO对照组,Western blot检测ERK5磷酸化水平,RT-qPCR检测两组IL-1β、TNF-αmRNA相对表达量。结果:LPS组IL-1β、TNF-αmRNA相对表达水平及蛋白表达水平均高于PBS组(P<0.05);lncRNA MIAT在LPS组相对表达量显著高于PBS组,且主要表达于细胞核;lncRNA MIAT在KD组显著低于NC组,其在J774A.1细胞的敲低效率为43%。KD组IL-1β、TNF-αmRNA相对表达量显著高于NC组;KD组ERK5磷酸化程度显著高于NC组,而NF-κB、STAT3、ERK1/2磷酸化水平差异无统计学意义;抑制剂组EKR5磷酸化显著低于DMSO对照组,且抑制剂组的IL-1β、TNF-αmRNA相对表达水平显著低于DMSO对照组。结论:lncRNA MIAT可能通过抑制ERK5磷酸化减少IL-1β、TNF-α合成,从而抑制巨噬细胞炎症反应。Objective:To explore role of long non-coding RNA myocardial infarction transcript(lncRNA MIAT)in macrophages inflammation and its possible inflammatory pathway.Methods:J774A.1 murine cells were cultured and stimulated by lipopolysaccharide(LPS)for inflammatory response cell model(LPS group)while PBS for negative control(PBS group).mRNA and protein expressions of IL-1βand TNF-αin LPS group and PBS group were detected by real-time quantitative PCR(RT-qPCR)and ELISA.lncRNA MIAT overall expression levels and subcellular localization in LPS group and PBS group were detected by RT-qPCR;lncRNA MIAT was knocked-down by shRNA and divided into shMIAT knockdown group(KD)and shNC negative control(NC)group.lncRNA MIAT knockdown efficiency,IL-1βand TNF-αmRNA relative expression levels in two groups were detected by RT-qPCR;expression levels of NF-κB,ERK5 and several transcription factors in KD and NC groups were detected by Western blot;LPS stimulated J774A.1 cells were divided into inhibitor group by adding extracellular regulated protein kinase 5 specific inhibitor(ERK5-IN-2)and negative control by adding dimethyl sulfoxide(DMSO).Level of ERK5 phosphorylation was detected by Western blot,relative expression of IL-1βand TNF-αmRNA in inhibitor group and DMSO group were detected by RT-qPCR.Results:mRNA,protein expression levels of IL-1βand TNF-αin LPS group were both significantly higher than those in PBS group;relative expression of lncRNA MIAT in LPS stimulated group was significantly higher than in PBS group,and mainly expressed in nucleus;significance of lncRNA MIAT in KD group was lower than NC group,which knockdown efficiency was 43%.IL-1βand TNF-αmRNA relative expression levels in KD group were significantly higher than that in NC group;ERK5 phosphorylation level in KD group was significantly higher than that in NC group,but there were no significant differences in NF-κB,STAT3 and ERK1/2 phosphorylation levels between KD group and NC groups;EKR5 phosphorylation level in inhibitor group was significantly lower

关 键 词：长链非编码RNA心肌梗死转录本 巨噬细胞 炎症 细胞外调节蛋白激酶5 

分 类 号：R392.12[医药卫生—免疫学]