大鼠肺和鼻上皮的过敏性炎症受组织特异性miRNA表达的调控
2022/06/17
摘要
背景：过敏性哮喘和过敏性鼻炎分别是影响下气道和鼻黏膜的常见慢性炎症性疾病。一些报道显示这两种疾病经常同时发生，然而，确切的分子机制尚未描述。
目的：本研究旨在探讨在过敏性气道炎症动物模型中，小非编码RNA是否可能与哮喘和过敏性鼻炎同时发生有关。
方法：作为过敏性气道炎症的体内模型，我们使用经鼻内暴露于室内尘螨(HDM)的Brown Norway大鼠。通过组织学分析、总IgE浓度、嗜酸性粒细胞计数和iNOS基因表达的检测来证实炎症改变。使用TruSeq小RNA文库制备试剂盒对肺组织和鼻上皮进行小RNA测序，并使用Base-Space工具进行分析。采用qPCR对测序结果进行验证。为了评估hsa-miR-223-3p的功能作用，我们用特异性的LNA抑制剂转染正常人支气管上皮(NHBE)细胞，并用ELISA检测NF-kB磷酸化蛋白水平。采用SYBR Green的qPCR技术对NF-kB通路相关基因进行表达分析，并在DataAssist v3.01中进行分析。使用STATISTICA第13版进行统计分析。
结果：我们发现过敏大鼠肺中有9个miRNA基因差异表达。在鼻上皮中，只有rno-miR-184在接触HDM的动物中上调。qPCR验证证实过敏大鼠肺中只有rno-miR-223-3p的表达增加。在IL-13刺激的正常支气管上皮ALI细胞培养中，该miRNA的表达也增加，但在单层培养的细胞中，由于IL13RA1和IL13RA2的mRNA水平较低，该miRNA的表达没有增加。转染hsa-miR-223-3p inhibitor后，NHBE细胞磷酸化NF-kB蛋白量增加，MUC5AC、CCL24和TSLP基因表达量增加。
结论：这些发现表明，调节肺部和鼻腔上皮过敏性炎症的miRNAs对上下气道是特异性的。此外，我们的研究为hsa-miR-223-3p通过靶向NF-kB信号通路调控MUC5AC、CCL24和TSLP的表达提供了新的见解。综上所述，我们的研究表明，miR-223-3p是过敏性炎症的调节因子，可能用于开发新的靶向治疗哮喘的药物。
Allergic inflammation in lungs and nasal epithelium of rat model is regulated by tissue-specific miRNA expression
Langwinski W, Szczepankiewicz D, Narozna B, et al.
Abstract
BACKGROUND：Atopic asthma and allergic rhinitis are common chronic inflammatory diseases affecting lower airways and nasal mucosa, respectively. Several reports demonstrated frequent co-occurrence of these two diseases, however, the exact molecular mechanism has not been described.
OBJECTIVE：The present study aimed to investigate if small non-coding RNA might be responsible for the co-occurrence of asthma and allergic rhinitis in an animal model of allergic airway inflammation.
METHODS：As an in vivo model of allergic airway inflammation, we used Brown Norway rats exposed intranasally to house dust mite (HDM). Histological analysis, total IgE concentration, eosinophil counts and iNOS gene expression were determined to confirm inflammatory changes. Small RNA sequencing in the lung tissue and nasal epithelium was performed with TruSeq Small RNA Library Preparation Kit and analyzed using the Base-Space tool. Validation of sequencing results was performed using qPCR. To assess the functional role of hsa-miR-223-3p, we transfected normal human bronchial epithelial (NHBE) cells with specific LNA-inhibitor and measured phosphorylated protein level of NF-kB with ELISA. Expression analysis of NF-kB pathway-related genes was performed using qPCR with SYBR Green and analyzed in DataAssist v3.01. Statistical analysis were done with STATISTICA version 13.
RESULTS：We found 9 miRNA genes differentially expressed in the lungs of allergic rats. In nasal epithelium, only rno-miR-184 was upregulated in animals exposed to HDM. Validation with qPCR confirmed increased expression only for rno-miR-223-3p in the lungs from allergic rats. The expression of this miRNA was also increased in normal bronchial epithelial ALI cell culture stimulated with IL-13, but not in cells cultured in monolayer due to the low mRNA level of IL13RA1 and IL13RA2. Transfecting NHBE cells with hsa-miR-223-3p inhibitor increased the amount of phosphorylated NF-kB protein level and expression of MUC5AC, CCL24 and TSLP genes.
CONCLUSIONS：These findings suggest that miRNAs that regulate allergic inflammation in the lungs and nasal epithelium are specific for upper and lower airways. Furthermore, our study provides new insight on the role of hsa-miR-223-3p, that via targeting NF-kB signaling pathway, regulates the expression of MUC5AC, CCL24 and TSLP. Taken together, our study suggests that miR-223-3p is a regulator of allergic inflammation and could potentially be used to develop novel and targeted therapy for asthma.
背景：过敏性哮喘和过敏性鼻炎分别是影响下气道和鼻黏膜的常见慢性炎症性疾病。一些报道显示这两种疾病经常同时发生，然而，确切的分子机制尚未描述。
目的：本研究旨在探讨在过敏性气道炎症动物模型中，小非编码RNA是否可能与哮喘和过敏性鼻炎同时发生有关。
方法：作为过敏性气道炎症的体内模型，我们使用经鼻内暴露于室内尘螨(HDM)的Brown Norway大鼠。通过组织学分析、总IgE浓度、嗜酸性粒细胞计数和iNOS基因表达的检测来证实炎症改变。使用TruSeq小RNA文库制备试剂盒对肺组织和鼻上皮进行小RNA测序，并使用Base-Space工具进行分析。采用qPCR对测序结果进行验证。为了评估hsa-miR-223-3p的功能作用，我们用特异性的LNA抑制剂转染正常人支气管上皮(NHBE)细胞，并用ELISA检测NF-kB磷酸化蛋白水平。采用SYBR Green的qPCR技术对NF-kB通路相关基因进行表达分析，并在DataAssist v3.01中进行分析。使用STATISTICA第13版进行统计分析。
结果：我们发现过敏大鼠肺中有9个miRNA基因差异表达。在鼻上皮中，只有rno-miR-184在接触HDM的动物中上调。qPCR验证证实过敏大鼠肺中只有rno-miR-223-3p的表达增加。在IL-13刺激的正常支气管上皮ALI细胞培养中，该miRNA的表达也增加，但在单层培养的细胞中，由于IL13RA1和IL13RA2的mRNA水平较低，该miRNA的表达没有增加。转染hsa-miR-223-3p inhibitor后，NHBE细胞磷酸化NF-kB蛋白量增加，MUC5AC、CCL24和TSLP基因表达量增加。
结论：这些发现表明，调节肺部和鼻腔上皮过敏性炎症的miRNAs对上下气道是特异性的。此外，我们的研究为hsa-miR-223-3p通过靶向NF-kB信号通路调控MUC5AC、CCL24和TSLP的表达提供了新的见解。综上所述，我们的研究表明，miR-223-3p是过敏性炎症的调节因子，可能用于开发新的靶向治疗哮喘的药物。
（中日友好医院呼吸与危重症医学科 李春晓 摘译 林江涛 审校）
（Molecular Immunology, 2022, 147: 115-125. doi: 10.1016/j.molimm.2022.04.017）
（Molecular Immunology, 2022, 147: 115-125. doi: 10.1016/j.molimm.2022.04.017）
Allergic inflammation in lungs and nasal epithelium of rat model is regulated by tissue-specific miRNA expression
Langwinski W, Szczepankiewicz D, Narozna B, et al.
Abstract
BACKGROUND：Atopic asthma and allergic rhinitis are common chronic inflammatory diseases affecting lower airways and nasal mucosa, respectively. Several reports demonstrated frequent co-occurrence of these two diseases, however, the exact molecular mechanism has not been described.
OBJECTIVE：The present study aimed to investigate if small non-coding RNA might be responsible for the co-occurrence of asthma and allergic rhinitis in an animal model of allergic airway inflammation.
METHODS：As an in vivo model of allergic airway inflammation, we used Brown Norway rats exposed intranasally to house dust mite (HDM). Histological analysis, total IgE concentration, eosinophil counts and iNOS gene expression were determined to confirm inflammatory changes. Small RNA sequencing in the lung tissue and nasal epithelium was performed with TruSeq Small RNA Library Preparation Kit and analyzed using the Base-Space tool. Validation of sequencing results was performed using qPCR. To assess the functional role of hsa-miR-223-3p, we transfected normal human bronchial epithelial (NHBE) cells with specific LNA-inhibitor and measured phosphorylated protein level of NF-kB with ELISA. Expression analysis of NF-kB pathway-related genes was performed using qPCR with SYBR Green and analyzed in DataAssist v3.01. Statistical analysis were done with STATISTICA version 13.
RESULTS：We found 9 miRNA genes differentially expressed in the lungs of allergic rats. In nasal epithelium, only rno-miR-184 was upregulated in animals exposed to HDM. Validation with qPCR confirmed increased expression only for rno-miR-223-3p in the lungs from allergic rats. The expression of this miRNA was also increased in normal bronchial epithelial ALI cell culture stimulated with IL-13, but not in cells cultured in monolayer due to the low mRNA level of IL13RA1 and IL13RA2. Transfecting NHBE cells with hsa-miR-223-3p inhibitor increased the amount of phosphorylated NF-kB protein level and expression of MUC5AC, CCL24 and TSLP genes.
CONCLUSIONS：These findings suggest that miRNAs that regulate allergic inflammation in the lungs and nasal epithelium are specific for upper and lower airways. Furthermore, our study provides new insight on the role of hsa-miR-223-3p, that via targeting NF-kB signaling pathway, regulates the expression of MUC5AC, CCL24 and TSLP. Taken together, our study suggests that miR-223-3p is a regulator of allergic inflammation and could potentially be used to develop novel and targeted therapy for asthma.
上一篇：
乌本苷可调节高Th2型哮喘小鼠的气道重塑
下一篇：
重组多聚体犬变应原可防止以强烈TH2和TH17细胞反应为标志的哮喘模型的气道高反应性
