摘要
[背景]铍致肺纤维化发病机制未明且没有特效治疗方法,微小RNA(miRNA)在铍致肺纤维化进程中或可发挥作用。[目的]构建微小RNA-21(miR-21)干扰细胞系,探讨miR-21对硫酸铍(BeSO_(4))所致人肺腺癌肺泡基底上皮细胞(A549细胞)纤维化的影响以及潜在的作用机制。[方法]通过在线数据库miRBase预测miR-21靶基因并通过双荧光素酶报告基因实验验证。用miR-21干扰慢病毒转染A549细胞后,用嘌呤霉素筛选出稳定细胞系。使用BeSO_(4)染毒A549细胞建立肺纤维化体外模型,BeSO_(4)浓度为10μmol·L^(-1),染毒时间为48 h。将细胞分为对照组、模型组、miR-21干扰组和miR-21干扰对照组。采用实时荧光定量PCR法检测miR-21 mRNA相对表达水平。采用蛋白免疫印迹检测TGF-β1/Smads通路相关蛋白[Smad2、Smad3、p-Smad2、p-Smad3、Smad7和转化生长因子-β1(TGF-β1)]表达水平,肌成纤维细胞标志物α-平滑肌肌动蛋白(α-SMA)和细胞外基质主要成分Ⅰ型胶原蛋白(COL-Ⅰ)和Ⅲ型胶原蛋白(COL-Ⅲ)的蛋白相对表达水平。[结果]miRBase预测miR-21与Smad7有结合点位,双荧光素酶报告基因实验结果显示miR-21靶基因为Smad7。miR-21干扰A549细胞系构建成功。与对照组相比,模型组miR-21mRNA相对表达量升高了97.57%,Smad7蛋白相对表达量下降了15.48%,Smad2、Smad3、p-Smad2、p-Smad3、TGF-β1、α-SMA、COL-Ⅰ和COL-Ⅲ的蛋白相对表达量分别升高了13.55%、35.72%、18.35%、35.75%、25.52%、31.58%、24.61%和11.66%,差异均具有统计学意义(P<0.05)。相较于干扰对照组,干扰组miR-21 mRNA相对表达水平降低28.96%,Smad7蛋白相对表达量升高了19.07%,Smad2、Smad3、p-Smad2、p-Smad3、TGF-β1、α-SMA、COL-Ⅰ和COL-Ⅲ的蛋白相对表达量降低了8.01%、19.95%、14.56%、19.37%、11.95%、10.96%、18.81%和31.36%,差异均具有统计学意义(P<0.05)。模型组与干扰对照组的各基因及蛋白表达水平差异无统计学意义(P>0.05)。[结论]在铍
[Background]The pathogenesis of beryllium-induced pulmonary fibrosis is unknown and there is no specific treatment for the disease as yet.MicroRNA(miRNA)may play a role in the process of beryllium-induced pulmonary fibrosis.[Objective]To construct a microRNA-21(miR-21)interfering cell line,and to investigate the effect of miR-21 on beryllium sulfate(BeSO_(4))-induced fibrosis in human lung adenocarcinoma alveolar basal epithelial cells(A549 cells)and its potential mechanism.[Methods]The miR-21 target genes were predicted by the online database miRBase and verified by experiments using dual luciferase reporter gene.After transfecting A549 with miR-21 interference lentivirus,puromycin was used to select a stable cell line.An in vitro model of pulmonary fibrosis was established using BeSO_(4)infecting A549 cells with a concentration of 10μmol·L^(-1)and an exposure time of 48 h.Then the treated cells were divided into control group,model group,miR-21 interference group,and miR-21 interference control group.Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the relative expression level of miR-21 gene.Western blotting was used to detect the relative expression levels of TGF-β1/Smads pathway related proteins[Smad2,Smad3,p-Smad2,p-Smad3,Smad7,and transforming growth factor-β1(TGF-β1)],myofibrosis cell markerα-smooth muscle actin(α-SMA),andextracellular matrix collagen-I(COL-I)and collagen-Ⅲ(COL-Ⅲ).[Results]The miRBase predicted that miR-21 had a binding site with Smad7,and the results of the dual luciferase reporter gene experiment showed that the target gene of miR-21 was Smad7.The construction of miR-21 interfered with A549 cell line was successful.Compared with the control group,the relative expression of miR-21 gene in the model group increased by 97.57%;the relative expression of Smad7 protein in the model group decreased by 15.48%;the relative protein expression of Smad2,Smad3,p-Smad2,pSmad3,TGF-β1,α-SMA,COL-I,and COL-Ⅲincreased by 13.55%,35.72%,18.35%,35.75%,25.52%,31.58%,24.61%,and
作者
戚发秋
陈小惠
刘红娅
赵枫
付有娟
关素珍
王凯
QI Faqiu;CHEN Xiaohui;LIU Hongya;ZHAO feng;FU Youjuan;GUAN Suzhen;WANG Kai(School of Public Health and Management/Ningxia Key Laboratory of Environmental Factors and Chronic Disease Control,Ningxia Medical University,Yinchuan,Ningxia 750004,China)
出处
《环境与职业医学》
CAS
CSCD
北大核心
2022年第2期206-211,共6页
Journal of Environmental and Occupational Medicine
基金
宁夏自然科学基金(2019 AAC03085)
