戚发秋, 陈小惠, 刘红娅, 赵枫, 付有娟, 关素珍, 王凯. 硫酸铍诱导A549细胞纤维化中miR-21靶向调控Smad7的机制[J]. 环境与职业医学, 2022, 39(2): 206-211. DOI: 10.11836/JEOM21312
引用本文: 戚发秋, 陈小惠, 刘红娅, 赵枫, 付有娟, 关素珍, 王凯. 硫酸铍诱导A549细胞纤维化中miR-21靶向调控Smad7的机制[J]. 环境与职业医学, 2022, 39(2): 206-211. DOI: 10.11836/JEOM21312
QI Faqiu, CHEN Xiaohui, LIU Hongya, ZHAO feng, FU Youjuan, GUAN Suzhen, WANG Kai. Mechanism of miR-21 targeting Smad7 in pulmonary fibrosis of A549 cells induced by beryllium sulfate[J]. Journal of Environmental and Occupational Medicine, 2022, 39(2): 206-211. DOI: 10.11836/JEOM21312
Citation: QI Faqiu, CHEN Xiaohui, LIU Hongya, ZHAO feng, FU Youjuan, GUAN Suzhen, WANG Kai. Mechanism of miR-21 targeting Smad7 in pulmonary fibrosis of A549 cells induced by beryllium sulfate[J]. Journal of Environmental and Occupational Medicine, 2022, 39(2): 206-211. DOI: 10.11836/JEOM21312

硫酸铍诱导A549细胞纤维化中miR-21靶向调控Smad7的机制

Mechanism of miR-21 targeting Smad7 in pulmonary fibrosis of A549 cells induced by beryllium sulfate

  • 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(BeSO4)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BeSO4染毒A549细胞建立肺纤维化体外模型,BeSO4浓度为10 μmol·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-21Smad7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Smad7miR-21干扰A549细胞系构建成功。与对照组相比,模型组miR-21 mRNA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 在铍化合物诱导的肺纤维化体外模型中,miR-21可能通过靶向Smad7调控TGF-β1/Smad信号通路促进了纤维化的发生。

     

    Abstract: 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 (BeSO4)-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-21interference lentivirus, puromycin was used to select a stable cell line. An in vitro model of pulmonary fibrosis was established using BeSO4 infecting A549 cells with a concentration of 10 μmol·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 marker α-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, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ increased by 13.55%, 35.72%, 18.35%, 35.75%, 25.52%, 31.58%, 24.61%, and 11.66% respectively (P<0.05). Compared with the interference control group, themiR-21 gene expression level in the interference group decreased by 28.96%; the relative expression of Smad7 protein increased by 19.07%; the relative protein expression of Smad2, Smad3, p-Smad2, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ decreased by 8.01%, 19.95%, 14.56%, 19.37%, 11.95%, 10.96%, 18.81%, and 31.36% repectively (P<0.05). There was no statistically significant difference in the gene abd protein expression levels of each gene between the model group and the interference control group (P>0.05).

    Conclusion In an in vitro model of pulmonary fibrosis induced by beryllium compounds, miR-21 may promote fibrosis by targeting Smad7 to regulate the TGF-β1/Smad signaling pathway.

     

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