miR-23b-5p通过靶向下调FN1缓解支气管哮喘气道重塑

miR-23b-5p relieves airway remodeling in bronchial asthma through targeted downregulation of FN1

目的探讨miR-23b-5p靶向下调纤维连接蛋白1(FN1)对支气管哮喘(简称哮喘)气道重塑的影响。方法将BALB/c小鼠随机分为正常对照组和哮喘组,每组6只。Masson染色观察气道壁周围胶原沉积水平。ELISA测定白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β1(TGF-β1)、血管内皮生长因子(VEGF)水平。实时荧光定量PCR(qRT-PCR)检测肺组织中miR-23b-5p表达水平。免疫组织化学染色、Western blotting检测FN1、α-平滑肌肌动蛋白(α-SMA)在肺组织中的表达。TargetScan网站预测miR-23b-5p靶基因,双荧光素酶报告实验验证。转染miR-23b-5p模拟物和抑制剂于人支气管平滑肌细胞(HBSMC)后,Western blotting检测FN1表达水平。结果与正常对照组相比,哮喘组小鼠气道上皮下胶原沉积,IL-6、TNF-α、TGF-β1、VEGF表达显著增高(P<0.001),miR-23b-5p表达显著降低(P<0.05)。免疫组织化学染色、Western blotting结果显示,FN1、α-SMA在哮喘组小鼠肺组织中的表达显著升高(P<0.05)。FN1受miR-23b-5p靶向调控,并且与miR-NC组相比,转染miR-23b-5p模拟物后FN1表达水平显著下调(P<0.05),而转染miR-23b-5p抑制剂后FN1表达水平显著升高(P<0.01)。结论miR-23b-5p可通过靶向下调FN1,缓解哮喘气道重塑。

Objective To investigate the effects of miR-23b-5p on airway remodeling in bronchial asthma through targeted downregulation of fibronectin 1(FN1).Methods BALB/c mice were randomly divided into a control or an asthma group(n=6 mice per group).Collagen deposition in the airway walls were detected using Masson staining.The levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),transforming growth factor-β1(TGF-β1),and vascular endothelial growth factor(VEGF)were determined using ELISA.Realtime fluorescence quantitative PCR(qRT-PCR)was performed to detect miR-23b-5p expression levels in lung tissues.Immunohistochemical staining and Western blotting were performed to detect the expression of FN1 andα-smooth muscle actin(α-SMA)in lung tissues.miR-23b-5p target genes were predicted using the TargetScan website,and validated using dual luciferase reporter assay.Western blotting was performed to detect the FN1 expression levels after transfection of miR-23b-5p mimic and inhibitor in human bronchial smooth muscle cells.Results Compared with the control group,mice in the asthma group had subepithelial collagen deposition in their airway,significantly higher levels of IL-6,TNF-α,TGF-β1,and VEGF(P<0.001),and significantly lower levels of miR-23b-5p(P<0.05).Immunohistochemical staining and Western blotting showed that the expression of FN1 andα-SMA significantly increased in the asthma group(P<0.05).FN1 was directly regulated by miR-23b-5p.Compared with the miR-NC group,FN1 expression levels were significantly down-regulated after transfection with miR-23b-5p mimic(P<0.05)and significantly increased after transfection with miR-23b-5p inhibitor(P<0.01).Conclusion miR-23b-5p relieves airway remodeling in bronchial asthma through the downregulation of FN1.