温石棉染毒肺上皮细胞的miRNA表达谱分析  

Expression profiling of miRNAs in chrysotile-exposed lung epithelial cells

作　　者：何佳睿 宋娟 王玉君 张旭 杨洁 霍婷婷[5] 董发勤[5] 邓建军 HE Jiarui;SONGJuan;WANG Yujun;ZHANG Xu;YANG Jie;HUO Tingting;DONG Faqin;DENG Jianjun(School of Laboratory Medicine,Chengdu Medical College,Chengdu,Sichuan 610500,China;Santai County People’s Hospital,Mianyang,Sichuan 621100,China;North Sichuan Medical College,Nanchong,Sichuan 637000,China;Mianyang 404 Hospital,Mianyang,Sichuan 621000,China;Southwest University of Science and Technology,Mianyang,Sichuan 621010,China)

机构地区：[1]成都医学院检验医学院,四川成都610500 [2]三台县人民医院,四川绵阳621100 [3]川北医学院,四川南充637000 [4]绵阳四〇四医院,四川绵阳621000 [5]西南科技大学,四川绵阳621010

出　　处：《环境与职业医学》2024年第11期1277-1282,1289,共7页Journal of Environmental and Occupational Medicine

基　　金：四川省自然科学基金项目(2023NSFSC0650);四川省医学青年创新科研课题计划项目(Q23073)。

摘　　要：[背景]温石棉被广泛应用于建筑、工业等领域。研究显示其易导致职业人群肺纤维化,但微小RNA(miRNA)参与温石棉致肺纤维化的研究较少,具体机制尚不明确。[目的]采用二代测序技术分析温石棉染毒对人肺上皮细胞(BEAS-2B细胞)miRNA表达谱的影响,探讨差异表达的miRNA及相关信号通路的变化,寻找温石棉诱导肺纤维化的潜在靶点及分子机制。[方法]本研究用激光粒度分析仪和X射线衍射仪对温石棉进行了粒径大小和物相分析,并对BEAS-2B细胞进行不同时间(12、24和48 h)、不同剂量(0、50、100和200μg·mL^(-1))的温石棉染毒。用细胞活力检测试剂盒(CCK8)检测细胞存活率。通过蛋白免疫印迹(WB)检测200μg·mL^(-1)温石棉染毒BEAS-2B细胞24 h后纤维连接蛋白(Fibronectin)、胶原蛋白Ⅰ(Collagen-Ⅰ)和α-平滑肌肌动蛋白(α-SMA)的表达水平。二代测序技术分析温石棉染毒后与对照组的样本相关性和miRNA表达谱的变化。预测差异miRNA的靶基因并对其进行基因功能注释(GO)分析和京都基因与基因组百科全书(KEGG)通路富集分析。[结果]本研究所用温石棉粉尘平均粒径为3.58μm,X射线衍射分析结果均为温石棉特征峰。与对照组比较,温石棉随浓度和染毒时间的增加而逐渐抑制BEAS-2B细胞存活率(P<0.01)。50、100和200μg·mL^(-1)温石棉染毒细胞12 h后的存活率分别为83.88%±1.86%、78.07%±3.97%、71.95%±2.99%;染毒24 h后的存活率分别为77.41%±1.58%、69.57%±2.23%、62.79%±3.65%;染毒48 h后的存活率分别为74.31%±4.93%、65.84%±2.71%、52.74%±6.31%。200μg·mL^(-1)温石棉染毒BEAS-2B细胞后Fibronectin、Collagen-Ⅰ和α-SMA蛋白表达水平升高(P<0.05)。温石棉实验组和对照组主成分分析显示两组样本成分存在差异,共筛选出163个差异miRNA,其中上调79个,下调84个。GO分析显示差异miRNA主要与RNA聚合酶Ⅱ的转录调控、DNA模板转录调控、细胞分化、蛋白磷[Background]Chrysotile is widely used in construction and industry.Research has shown that it is associated with lung fibrosis in occupational groups,but the involvement of microRNAs(miRNAs)in chrysotile-induced lung fibrosis hasbeen less well studied,and the specific mechanism is still unclear.[Objective]Using next-generation sequencing technology to analyze the effects of chrysotile exposure on the miRNAs expression profilesof human lung epithelial cells(BEAS-2B cells),to explore the variations of differentially expressed miRNAs and related signaling pathways,and to identify potential targets and molecular mechanisms of chrysotile-induced lung fibrosis.[Methods]Chrysotile was analyzed with a laser particle size analyzer and an X-ray diffractometer for particle size and physical phase.BEAS-2B cells were exposed to chrysotile for designed time sessions(12,24,and 48 h)and doses(0,50,100,and 200μg·mL^(-1)).Cell viabilitywas detected with a cell viability assay kit(CCK8);expression levels of Fibronectin,Collagen-Ⅰ,andα-smooth muscle actin(α-SMA)weredetected by Western blot after exposure to 200μg·mL^(-1) chrysotile for 24 h.Sample correlation and changes in miRNAs expression profilesbetween the chrysotile-exposed and the control groups were analyzed by next-generation sequencing technology.The target genes ofdifferentially expressed miRNAs were predicted and subjected to Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes andGenomes(KEGG)pathway enrichment analysis.[Results]The average particle size of the chrysotile dust sample used in this study was 3.58μm,and the results of X-ray diffraction analysisconfirmed the characteristic peaks of chrysotile.Compared with the control group,the chrysotile gradually inhibited the survival rate ofBEAS-2B cells with increasing concentration and exposure time(P<0.01).The survival rates of the 50,100,and 200μg·mL^(-1) chrysotile-exposedcells after 12 h exposure were 83.88%±1.86%,78.07%±3.97%,and 71.95%±2.99%,respectively;the survival rates after 24 h exposurew

关 键 词：温石棉 肺纤维化 微小RNA BEAS-2B细胞 

分 类 号：R11[医药卫生—公共卫生与预防医学]