Abstract：
Allergic asthma is a growing burden on national public health services due to its high prevalence. The aim of this experiment was to investigate whether miR-26a-5p affects cellular fibrosis and thus airway remodeling in asthmatic mice through the regulation of target genes.
Screening for differentially expressed miRNAs in asthma model mice was carried out by constructing a mouse model of allergic asthma. qRT-PCR was performed to determine candidate miRNAs in each group of bronchial tissues. Western blot detection of the expression levels of predicted candidate target genes in each group of bronchial tissues was conducted. A dual luciferase assay was performed to validate the binding of miR-26a-5p to target genes. Fibronectin, a marker of cellular fibrosis, was detected via flow cytometry. CCK8 and BrdU staining were used to detect the proliferation ability of each group of cells.
miR-26a-5p is able to target and bind to ABL2 3\'-UTR, MMP16 3\'-UTR and PDE7A 3\'-UTR sequences. After interference with miR-26a-5p, improved bronchial histopathology and reduced peribronchial collagen deposition were found. Compared with the model group, interference with miR-26a-5p reduced lung fibrosis, decreased fibroblasts and increased apoptosis in mouse bronchial tissues; overexpression of miR-26a-5p decreased apoptosis in mouse bronchial tissues. Compared with the model group, the serum levels of IL-4, IL-5, IL-13 and I IFN-γ were decreased in the miR-26a-5p inhibitor group and increased in the miR-26a-5p mimic group. The immunohistochemical results showed that the expression of ABL2, MMP16 and PDE7A was significantly reduced after intervention with miR-26a-5p. Compared with the model group, the apoptosis rate of cells in the miR-26a-5p inhibitor group of the allergic asthma model was upregulated, the levels of IL-4, IL-5, IL-13, IFN-γ and ROS were decreased, the expression of the miRNA and proteins of ABL2, MMP16 and PDE7A was decreased, the expression of LC3A and P62 was significantly increased and the expression of LC3B, Beclin1, Atg5 and fibrosis markers collagen I and α-SMA was decreased.
miR-26a-5p affects cellular fibrosis and thus airway remodeling in asthmatic mice by regulating target genes.
Screening for differentially expressed miRNAs in asthma model mice was carried out by constructing a mouse model of allergic asthma. qRT-PCR was performed to determine candidate miRNAs in each group of bronchial tissues. Western blot detection of the expression levels of predicted candidate target genes in each group of bronchial tissues was conducted. A dual luciferase assay was performed to validate the binding of miR-26a-5p to target genes. Fibronectin, a marker of cellular fibrosis, was detected via flow cytometry. CCK8 and BrdU staining were used to detect the proliferation ability of each group of cells.
miR-26a-5p is able to target and bind to ABL2 3\'-UTR, MMP16 3\'-UTR and PDE7A 3\'-UTR sequences. After interference with miR-26a-5p, improved bronchial histopathology and reduced peribronchial collagen deposition were found. Compared with the model group, interference with miR-26a-5p reduced lung fibrosis, decreased fibroblasts and increased apoptosis in mouse bronchial tissues; overexpression of miR-26a-5p decreased apoptosis in mouse bronchial tissues. Compared with the model group, the serum levels of IL-4, IL-5, IL-13 and I IFN-γ were decreased in the miR-26a-5p inhibitor group and increased in the miR-26a-5p mimic group. The immunohistochemical results showed that the expression of ABL2, MMP16 and PDE7A was significantly reduced after intervention with miR-26a-5p. Compared with the model group, the apoptosis rate of cells in the miR-26a-5p inhibitor group of the allergic asthma model was upregulated, the levels of IL-4, IL-5, IL-13, IFN-γ and ROS were decreased, the expression of the miRNA and proteins of ABL2, MMP16 and PDE7A was decreased, the expression of LC3A and P62 was significantly increased and the expression of LC3B, Beclin1, Atg5 and fibrosis markers collagen I and α-SMA was decreased.
miR-26a-5p affects cellular fibrosis and thus airway remodeling in asthmatic mice by regulating target genes.
摘要：
目的：过敏性哮喘由于其发病率较高，对国家公共卫生服务造成了越来越大的负担。该实验的目的是研究miR-26a-5p是否通过调节靶基因影响哮喘小鼠的细胞纤维化并因此影响气道重塑。
方法：通过构建过敏性哮喘小鼠模型，筛选哮喘模型小鼠中差异表达的miRNA。进行qRT-PCR以确定每组支气管组织中的候选miRNA。Westernblot检测各组支气管组织中预测的候选靶基因的表达水平。进行双荧光素酶测定以验证miR-26a-5p与靶基因的结合。纤连蛋白,细胞纤维化的标志，通过流式细胞术检测。CCK8和BrdU染色检测各组细胞的增殖才能。
结果：miR-26a-5p能够靶向并结合ABL23'-UTR，MMP163'-UTR和PDE7A3'-UTR序列。干扰miR-26a-5p后，发现支气管组织病理学改善和支气管周围胶原沉积减少。与模型组相比，干扰miR-26a-5p减少肺纤维化，小鼠支气管组织中的成纤维细胞减少和细胞凋亡增加;miR-26a-5p的过表达减少小鼠支气管组织中的细胞凋亡。与模型组相比，血清IL-4,IL-5,IL-13和IIFN-γ水平在miR-26a-5p抑制剂组中降低,在miR-26a-5p模拟组中升高.免疫组化成果显示,miR-26a-5p干预后,ABL2、MMP16和PDE7A的表达显著降低。与模型组相比，miR-26a-5p抑制剂组细胞凋亡率上调,IL-4、IL-5、IL-13、IFN-γ和ROS水平降低,ABL2、MMP16和PDE7A的miRNA和蛋白的表达降低,LC3A和P62的表达显著增加,Beclin1、Atg5和纤维化标志物胶原I和α-SMA降低。
结论：miR-26a-5p通过调节靶基因影响哮喘小鼠的细胞纤维化和气道重塑。
方法：通过构建过敏性哮喘小鼠模型，筛选哮喘模型小鼠中差异表达的miRNA。进行qRT-PCR以确定每组支气管组织中的候选miRNA。Westernblot检测各组支气管组织中预测的候选靶基因的表达水平。进行双荧光素酶测定以验证miR-26a-5p与靶基因的结合。纤连蛋白,细胞纤维化的标志，通过流式细胞术检测。CCK8和BrdU染色检测各组细胞的增殖才能。
结果：miR-26a-5p能够靶向并结合ABL23'-UTR，MMP163'-UTR和PDE7A3'-UTR序列。干扰miR-26a-5p后，发现支气管组织病理学改善和支气管周围胶原沉积减少。与模型组相比，干扰miR-26a-5p减少肺纤维化，小鼠支气管组织中的成纤维细胞减少和细胞凋亡增加;miR-26a-5p的过表达减少小鼠支气管组织中的细胞凋亡。与模型组相比，血清IL-4,IL-5,IL-13和IIFN-γ水平在miR-26a-5p抑制剂组中降低,在miR-26a-5p模拟组中升高.免疫组化成果显示,miR-26a-5p干预后,ABL2、MMP16和PDE7A的表达显著降低。与模型组相比，miR-26a-5p抑制剂组细胞凋亡率上调,IL-4、IL-5、IL-13、IFN-γ和ROS水平降低,ABL2、MMP16和PDE7A的miRNA和蛋白的表达降低,LC3A和P62的表达显著增加,Beclin1、Atg5和纤维化标志物胶原I和α-SMA降低。
结论：miR-26a-5p通过调节靶基因影响哮喘小鼠的细胞纤维化和气道重塑。
