miR-27b-3p对脓毒症肺内皮细胞YAP信号表达的调控机制研究  

miR-27b-3p in the regulation of the YAP signaling in the endothelial cells isolated from the sepsis mice

作　　者：黄爱华[1] 王旸 HUANG Aihua;WANG Yang(The Intensive Care Unit(ICU),Hangzhou Third People's Hospital,Hangzhou 310009,China;Department of Embryology,School of The Basic Medical Sciences,Wenzhou Medical University,Wenzhou,Zhejiang 325035,China)

机构地区：[1]杭州市第三人民医院急诊科,杭州310009 [2]温州医科大学基础医学院组胚教研室,温州325035

出　　处：《浙江中西医结合杂志》2021年第3期221-225,共5页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

摘　　要：目的探讨微小RNA 27b-3p(miR-27b-3p)对脓毒症模型小鼠肺内皮细胞Yes相关蛋白(YAP)信号的调控机制。方法将40只C57BL/6雄性小鼠按照随机数字表法分为四组,对照组、盲肠结扎穿孔(CLP)组、miRNA类似物(mimic NC)组、miR-27b-3p类似物(miR-27b-3p mimic)组。利用CLP法构建脓毒症模型小鼠,miR-27b-3p组小鼠通过尾静脉注射miR-27b-3p mimic,mimic NC组小鼠尾静脉注射mimic NC阴性对照物。分离小鼠原代肺内皮细胞,荧光定量PCR(qRT-PCR)检测细胞YAP、细胞间粘附分子(ICAM)、选择素(E-selectin)miR-27b-3p mRNA表达水平;数据库PITA预测调控YAP的miRNA;蛋白免疫印迹(Western blot)法检测细胞YAP、ICAM、E-selectin表达及p65活化水平;酶联免疫吸附试验(ELISA)检测细胞上清液肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)水平。结果与对照组比较,CLP组小鼠原代肺内皮细胞YAP、ICAM、E-selectin mRNA表达明显升高[YAP:(3.21±0.25)比(1.00±0.12);ICAM:(2.36±0.31)比(1.00±0.12);E-selectin:(1.98±0.11)比(1.00±0.12),P<0.05];miR-27b-3p表达明显下调[(0.31±0.15)比(1.00±0.11),P<0.05];数据库PITA预测miR-27b-3p与YAP mRNA 3'UTR序列存在6个完全互补配对碱基UGUGAA;Western blot结果显示,与对照组比较,CLP组YAP、ICAM、E-selectin等蛋白水平明显升高,同时p-p65磷酸化活化水平明显升高;与mimic NC组比较,miR-27b-3p mimic组YAP表达量明显下调,ICAM、E-selectin表达水平明显升高,同时p65磷酸化水平升高;ELISA结果显示,与对照组比较,CLP组小鼠内皮上清液TNF-α、IL-6水平升高[TNF-α:(4.23±0.35)pg/mL比(1.00±0.52)pg/mL;IL-6:(7.43±0.83)pg/mL比(1.00±0.52)pg/mL,P<0.05];与mimic NC组比较,miR-27b-3p mimic组小鼠内皮上清液TNF-α、IL-6的含量明显升高[TNF-α:(8.52±0.46)pg/mL比(4.15±0.24)pg/mL;IL-6:(10.16±0.62)pg/mL比(7.85±0.57)pg/mL,P<0.05]。结论脓毒症诱导内皮低表达miR-27b-3p,促进YAP在脓毒症内皮细胞进一步高表达,从而抑制负向反馈调控内皮炎症信号�Objective To investigate the role of miR-27b-3p in the regulation of the Yes-associated protein(YAP)signaling in the endothelial cells isolated from the sepsis mice.Methods A total of 40 male C57BL/6 mice were randomly divided into four groups,i.e.,the control,the cecal ligation and perforation(CLP),CLP plus miR-27b-3p negative control(NC),and CLP plus miR-27b-3p mimic groups.The mouse sepsis model was established by using CLP,while miR-27b-3p mimic or NC was injected into the tail vein of mice.After that,primary pulmonary endothelial cells(PPECs)were isolated.mRNA levels of Yap,ICAM,E-selectin and miR-27b-3p were detected using qRT-PCR.The miRNAs targeting YAP were predicted using PITA database.Protein levels of Yap,ICAM,E-selectin and phosphorylation of p65(p-p65)were detected using Western blot.Levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in the endothelial cell culture supernatants were detected using the ELISA.Results Compared with the control group,mRNA levels of Yap(3.21±0.25 vs.1.00±0.12;P<0.05),ICAM(2.36±0.31 vs.1.00±0.12;P<0.05),and E-selectin(1.98±0.11 vs.1.00±0.12;P<0.05)in PPECs isolated from the CLP mice were significantly upregulated,whereas miR-27b-3p level was significantly downregulated(0.31±0.15 vs.1.00±0.11;P<0.05).The database PITA predicted six fully complementary paired bases(5'-UGUGAA-3')of the miR-27b-3p in the YAP mRNA 3'-untranslated region.The Western blot data showed that levels of YAP,ICAM,E-selectin,and p-p65 proteins were significantly increased in the CLP group compared to the control group.Compared with the miR-27b-3p NC group,YAP protein was significantly downregulated,while ICAM,E-selectin and p-p65 levels were significantly increased in the miR-27b-3p group.The ELISA results revealed that TNF-αand IL-6 levels in the endothelial cell culture supernatant from the CLP mice were increased compared with that from the control group[TNF-α:(4.23±0.35)vs.(1.00±0.52)pg/mL;IL-6:(7.43±0.83)vs.(1.00±0.52)pg/mL;P<0.05].Compared with the miR-27b-3p NC group,

关 键 词：小鼠 脓毒症 miR-27b-3p Yes相关蛋白 炎症 

分 类 号：R459.7[医药卫生—急诊医学]