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目的:探讨microRNA-33a(miR-33a)在哮喘患儿及哮喘小鼠中的表达及其对气道黏液高分泌的作用。方法:RT-PCR检测哮喘患儿和健康儿童诱导痰,以及卵清蛋白(ovalbumin,OVA)诱导的哮喘小鼠和正常小鼠肺组织中miR-33a的表达;分别鼻滴miR-33a模拟物(miR-33a mimics)及其对照(mimics NC)至哮喘小鼠,ELISA检测各组小鼠支气管肺泡灌洗液(BALF)上清中IL-13、TNF-α及黏蛋白MUC5AC的表达,RTPCR和western blot检测小鼠肺组织MUC5AC和pendrin(SLC26A4)的表达;双荧光素酶报告法验证miRNA-33a的靶基因。结果:哮喘患儿诱导痰及哮喘小鼠BALF中miR-33a表达显著降低(P<0.05);哮喘小鼠BALF中嗜酸性粒细胞(Eosnophils,EOS)数目显著增加,IL-13、TNF-α和MUC5AC表达显著升高(P<0.05);miR-33a mimics滴入导致EOS数目减少,IL-13、TNF-α和MUC5AC表达显著降低(P<0.05),同时小鼠肺组织MUC5AC和pendrin显著下调(P<0.05);miR-33a mimics明显抑制pendrin(SLC26A4)野生型报告基因的荧光素酶活性(P<0.05);pendrin(SLC26A4)过表达显著上调miR-33a mimics转染细胞中MUC5AC的表达(P<0.05)。结论:miR-33a通过靶向pendrin抑制黏蛋白MUC5AC的表达,抑制支气管哮喘气道黏液高分泌。
Objective: To investigate the expression of microRNA-33a (miR-33a) in asthmatic children and asthmatic mice and its effect on airway mucus hypersecretion. Methods: The expression of miR-33a in induced sputum and lung tissue of asthmatic mice and normal mice induced by ovalbumin (OVA) were detected by RT-PCR. Nasal drop miR-33a mimics (miR-33a mimics) and its control (mimics NC) to asthmatic mice. The levels of IL-13, TNF-α and mucin MUC5AC in bronchoalveolar lavage fluid (BALF) And western blot were used to detect the expression of MUC5AC and pendrin (SLC26A4) in mouse lung tissue. The target gene of miRNA-33a was verified by dual luciferase reporter assay. Results: The expression of miR-33a in BALF was significantly decreased in asthmatic mice induced by sputum and asthma (P <0.05), the number of eosinophils (EOS) in asthmatic BALF was significantly increased, the levels of IL-13 and TNF-α (P <0.05). The number of EOS decreased, the expression of IL-13, TNF-α and MUC5AC significantly decreased (P <0.05), and MUC5AC and pendrin in lung tissue of mice were significantly decreased (P <0.05); miR-33a mimics significantly inhibited the luciferase activity of pendrin (SLC26A4) wild-type reporter gene (P <0.05); overexpression of pendrin (SLC26A4) significantly upregulated the expression of MUC5AC in miR-33a mimics transfected cells Expression (P <0.05). Conclusion: miR-33a inhibits the mucus hypersecretion of bronchial asthma by targeting pendrin to inhibit the expression of mucin MUC5AC.