摘要
目的探讨微小RNA(miR)-133b对烟草提取物(CSE)诱导的人小气道上皮细胞间充质转化的影响及其调控机制。方法基因表达数据库(GEO数据库)中搜索慢性阻塞性肺疾病患者气道上皮细胞miR的表达谱芯片,寻找差异表达的miR并用实时荧光定量聚合酶链反应法(qRT-PCR)验证;小气道上皮细胞(HSAEpiC)根据干预方式不同分为对照组、CSE组、CSE+miR-133b抑制剂转染组(抑制剂组),CSE+miR-133b抑制剂对照转染组(抑制剂对照组)。观察各组细胞形态变化,qRT-PCR法检测各组miR-133b、转化生长因子(TGF)-β1 mRNA、Smad2 mRNA、钙黏附蛋白E(E-cadherin)mRNA与波形蛋白(Vimentin)mRNA,酶联免疫吸附法(ELISA)及免疫印迹法(Western blot)检测细胞上清液及细胞E-cadherin与Vimentin蛋白水平。结果在GSE53519发现9个差异表达的miR,验证后发现miR-133b在HSAEpiC细胞模型中表达差异最为显著。CSE诱导HSAEpiC细胞形态改变,miR-133b抑制剂能部分逆转细胞形态变化。CSE诱导HSAEpiC细胞上皮表型标志物E-cadherin mRNA及蛋白表达减少、间质表型标志物Vimentin mRNA及蛋白表达增多(F=9.09、12.35、7.57、101.87,P=0.015、0.007、0.023、0.000);miR-133b抑制剂能部分逆转E-cadherin Vimentin mRNA及蛋白表达(F=40.59、27.74、15.87、20.42,P=0.000、0.001、0.004、0.002)。CSE诱导HSAEpiC细胞TGF-β1 mRNA、Smad mRNA表达增多,miR-133b抑制剂部分逆转TGF-β、mRNA、Smad mRNA的改变(F=17.25、64.15,P=0.003、0.000)。结论miR-133b可能通过TGF-β/Smad通路调控CSE诱导的HSAEpiC细胞上皮间充质转化。
Objective To investigate the effects of microRNA(miR)-133b on epithelial-mesenchymal transition(EM)of human small airway epithelial cells induced by cigarette smoke extracts(CSE)and its regulatory mechanisms.Methods The miR-expression profiles with microarray in airway epithelial cells of patients with chronic obstructive pulmonary disease were searched in the Gene Expression Omnibus(GEO)database,and the differentially expressed miRs were searched and verified by a real-time fluorescence quantitative method(qRT-PCR).Human small airway epithelial cells(HSAEpiC)were divided into the control group,the CSE group,the CSE+miR-133b inhibitor transfection group(inhibitor group)and the CSE+miR-133b inhibitor negative control transfection group(inhibitor control group)according to different intervention methods.Levels of miR-133b and mRNA levels of transforming growth factor(TGF)-β1,Smad2,E-cadherin and vimentin were detected by RT-PCR;Protein levels of E-cadherin and vimentin were detected by enzyme-linked immunosorbent assays(ELISA)and Western blotting.Results Nine differentially expressed miRs were found in GSE53519,with miR-133b showing the most significant differential in thee HSAEpiC cell model after verification.CSE induced morphological changes in HSAEpiC cells,and miR-133b inhibitors could partially reverse the morphological changes in cell mode.mRNA and protein expressions of E-cadherin were decreased and expression of Vimentin mRNA and protein were increared in CSE induced HSAEpiC cells(F=9.09、12.35、7.57、101.87,P=0.015、0.007、0.023、0.000);miR-133b inhibitors partally reversed the mRNA and protein expressions of E-cadherin and Vimentin(F=40.59、27.74、15.87、20.42,P=0.000、0.001、0.004、0.002).CSE induced incresed expression of TGF-β1 mRNA and Smad mRNA in HSAEpiC cells,and miR-133b inhibitors partially reversed the changes in TGF-β1 mRNA and Smad mRNA(F=17.25、64.15,P=0.003、0.000).Conclusions miR-133b may regulate CSE-related HSAEpiC cell EMT through the TGF-β1/Smad pathway.
作者
连宁芳
张舒怡
高少勇
吕晓婷
林其昌
Lian Ningfang;Zhang Shuyi;Gao Shaoyong;Lyu Xiaoting;Lin Qichang(Department of Pulmonary and Critical Care Medicine,the First Affiliated Hospital of Fujian Medical University,Fuzhou 350005,China)
出处
《中华老年医学杂志》
CAS
CSCD
北大核心
2020年第3期336-340,共5页
Chinese Journal of Geriatrics
基金
福建省卫生厅中青年骨干人才培养项目(2017-ZQN-43)。
关键词
肺疾病
慢性阻塞性
烟草
上皮细胞
Pulmonary disease,chronic obstructive
Tobacco
Epithelial cells
