摘要
目的探讨微小RNA(miR)-130a-3p减轻缺氧/复氧心肌微血管内皮细胞(CMECs)炎症的调控机制。方法分离培养CMECs,分别将miR-130a-3p模拟物(mimics)及其阴性对照(miR-NC)、硫氧还蛋白结合蛋白过表达载体(pc-TXNIP)及其对照(pcDNA3.1)转染细胞,细胞共分为6组:对照组、缺氧/复氧(H/R)组、mimic组(转染mimics)、miR-NC组(转染miR-NC)、pcDNA3.1组(转染mimics和pcDNA3.1)和pc-TXNIP组(转染mimics和pc-TXNIP),除对照组以外的其他细胞在转染后建立H/R模型(缺氧6 h后复氧4 h),再培养24 h。观察miR-130a-3p表达水平以及对细胞活力、乳酸脱氢酶(LDH)、白细胞介素(IL)-1β和TXNIP、NLRP3表达的影响。双荧光素酶报告基因实验验证miR-130a-3p和TXNIP之间的靶向关系。各组间差异比较采用方差分析。结果H/R组及miR-NC组细胞活力均低于对照组(0.39±0.03、0.35±0.04比1.00±0.01,t=6.753、7.248,P<0.05),H/R组及miR-NC组LDH水平均高于对照组[(69.43±7.37)U/L、(65.24±8.21)U/L比(25.07±2.46)U/L,t=8.526、7.983,P<0.05];mimics组细胞活力高于miR-NC组(0.68±0.07比0.35±0.04,t=6.273,P<0.05),mimics组LDH、IL-1β水平低于miR-NC组[(41.07±3.06)U/L比(65.24±8.21)U/L,(41.07±3.06)pg/ml比(65.24±8.21)pg/ml,t=4.257、6.693,P<0.05]。mimics组TXNIP和NLRP3蛋白相对表达量低于miR-NC组(2.64±0.34比4.59±0.38、3.67±0.49比6.35±0.58,t=7.273、6.928,P<0.05)。pc-TXNIP组细胞活力低于pcDNA3.1组(0.37±0.04比0.65±0.08,t=6.463,P<0.05),pc-TXNIP组LDH和IL-1β水平高于pcDNA3.1组[(62.24±6.47)U/L比(42.33±2.89)U/L、(55.24±6.47)pg/ml比(42.33±2.89)pg/ml,t=7.264、6.613,P<0.05],pc-TXNIP组TXNIP和NLRP3蛋白表达水平高于pcDNA3.1组(4.61±0.37比2.28±0.31、5.07±0.63比3.35±0.41,t=7.248、6.375,P<0.05)。采用双荧光素酶报告基因验证miR-130a-3p与TXNIP之间存在靶向关系。结论CMECs经H/R处理后miR-130a-3p表达下调,上调miR-130a-3p表达可减轻CMECs损伤和炎性反应,该保护作用与miR-130a-3p靶向调控TXNIP、减少NLRP3�
Objective To observe the regulatory mechanism of microRNA(miR)-130a-3p in reducing inflammation of myocardial microvascular endothelial cells(CMECs)induced by hypoxia/reoxygenation(H/R).Methods CMECs were isolated and cultured.The miR-130a-3p mimics and its negative control(miR-NC),pc-thioredoxin-interacting protein(pc-TXNIP)and its control(pcDNA3.1)were transfected into the CMECs,and H/R models(hypoxia 6 h and reoxygenation 4 h)were established.The effects of miR-130a-3p expression on cell viability,lactate dehydrogenase(LDH),interleukin(IL)-1β,and the expression of TXNIP and NOD-like receptor family,pyrin domain containing 3(NLRP3)were observed.Dual luciferase reporter assay was used to verify the targeting relationship between miR-130a-3p and TXNIP.Results The cell viability in H/R group and miR-NC group was lower than that in control group(0.39±0.03,0.35±0.04 vs.1.00±0.01,t=6.753,7.248,P<0.05).LDH levels in H/R and miR-NC groups were higher than in control group[(69.43±7.37),and(65.24±8.21)U/L vs.(25.07±2.46)U/L,t=8.526,7.983,P<0.05].Cell viability in mimics group was higher than in miR-NC group(0.68±0.07 vs.0.35±0.04,t=6.273,P<0.05).The levels of LDH and IL-1βwere lower in mimics group than in miR-NC group[(41.07±3.06)U/L vs.(65.24±8.21)U/L,(41.07±3.06)pg/ml vs.(65.24±8.21)pg/ml,t=4.257,6.693,P<0.05].The expression of TXNIP and NLRP3 protein in mimics group was lower than in miR-NC group(2.64±0.34 vs.4.59±0.38,3.67±0.49 vs.6.35±0.58,t=7.273,6.928,P<0.05).Cell viability in pc-TXNIP group was lower than in pcDNA3.1 group(0.37±0.04 vs.0.675±0.08,t=6.463,P<0.05).The levels of LDH and IL-1βin pc-TXNIP group were higher than in pcDNA3.1 group[(62.24±6.47)vs.(42.33±2.89)U/L,(55.24±6.47)vs.(42.33±2.89)pg/ml,t=7.264,6.613,P<0.05].The levels of TXNIP and NLRP3 protein in pc-TXNIP group were higher than in pcDNA3.1 group(4.61±0.37 vs.2.28±0.31,5.07±0.63 vs.3.35±0.41,t=7.248,6.375,P<0.05).The target relationship between miR-130a-3p and TXNIP was confirmed by dual luciferase reporter assay.Co
作者
周涛
高士豪
张伟
蒙国鑫
Zhou Tao;Gao Shihao;Zhang Wei;Meng Guoxin(Department of Cardiac Surgery,Guizhou Provincial People’s Hospital,Guiyang 550002,China)
出处
《中华实验外科杂志》
CAS
北大核心
2023年第1期83-86,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(81960050、82160056)
贵州省科技计划项目(科学技术基金)(黔科合基础[2020]1Y293号)
贵州省卫生健康委员会科学技术基金项目(gzwkj2021-193)
贵州省人民医院2019年度国家自然科学基金后补助基金(GPPH-NSFC-2019-24)。
关键词
微小RNA
内皮细胞
炎症
硫氧还蛋白结合蛋白
炎症小体
MicroRNA
Endothelial cells
Inflammation
Thioredoxin-interacting protein
NOD-like receptor family,pyrin domain containing 3
