摘要
目的 miRNA-155在支气管哮喘中的作用及其机制研究较少,文章拟探讨miRNA-155通过靶向Wnt5a调控转录激活子3(STAT3)信号通路在大鼠支气管哮喘发生发展中的作用。方法将SD大鼠按随机数字表法分为正常对照组、阴性对照组和miRNA-155表达抑制组,每组12只,正常对照组大鼠腹腔注射等渗盐水,阴性对照组大鼠在支气管哮喘模型建立后转染NC拮抗物;miRNA-155表达抑制组大鼠在支气管哮喘模型建立后转染miRNA-155拮抗物;分别于转染后7 d麻醉处死,采用HE染色法观察肺组织损伤情况;采用qRT-PCR法检测各组大鼠肺组织miRNA-155、Wnt5a mRNA表达水平;采用双荧光素酶报告基因实验验证miRNA-155与Wnt5a的靶向关系;采用Western blot法检测各组大鼠肺组织Wnt5a、STAT3、p-STAT3的蛋白表达水平;采用ELISA法分别检测肺组织匀浆IL-6、TNF-α表达水平。结果与正常对照组比较,阴性对照组大鼠肺功能指标FVC、FEV1及PEF均降低而FEV1/FVC升高(P<0.05),且miRNA-155表达水平升高,Wnt5a基因mRNA和蛋白表达水平均降低(P<0.05);与阴性对照组相比,miRNA-155表达抑制组大鼠肺功能均有不同程度的提高(P<0.05),miRNA-155表达水平降低(P<0.05),Wnt5a基因mRNA和蛋白表达水平均升高(P<0.05);双荧光素酶报告基因实验分析结果显示,miRNA-155和Wnt5a能够靶向结合;与正常对照组大鼠肺组织STAT3、p-STAT3的蛋白表达水平[(0.20±0.05)、(0.22±0.05)]相比,阴性对照组[(1.35±0.05)和(1.11±0.05)]和miRNA-155表达抑制组大鼠[(1.01±0.06)、(0.52±0.07)]均升高(P<0.05),miRNA-155表达抑制组大鼠肺组织STAT3、p-STAT3蛋白表达水平较阴性对照组降低(P<0.05);与正常对照组大鼠肺组织IL-6、TNF-α表达水平[(95.92±8.78)、(88.99±8.12)pg/mL]相比,阴性对照组[(206.11±18.63)、(177.54±13.12)pg/mL]和miRNA-155表达抑制组[(156.17±9.55)、(131.94±9.25)pg/mL]均升高(P<0.05);与阴性对照组相比,miRNA-155表达抑制组大鼠肺组织IL-6、TNF-α表达水平降低(P<0.05)。结论 miRNA-155通过靶向Wnt5a激活STAT3信号通路促进支气管哮喘的发生发展。
Objective miRNA-155 is involved in a variety of biological processes and plays an important role in the pathogenesis of different allergic diseases and atopic dermatitis. However, the role of miRNA-155 in bronchial asthma and its mechanism are rarely studied. Therefore, this study was to explore the role of miRNA-155 in the occurrence and development of bronchial asthma in rats by targeting Wnt5 a to regulate the STAT3 signaling pathway. Methods SD rats were randomly divided into normal control group, negative control group, and miRNA-155 expression inhibition group, each with 12 rats. The normal control group was intraperitoneally injected with isotonic saline;the negative control group was transfected with NC antagomir after the establishment of the bronchial asthma model;the miRNA-155 expression inhibition group was transfected with miRNA-155 antagomir after the model establishment. Seven days after transfection, the rats were anesthetized and sacrificed. The lung injury was observed by HE staining. The expression levels of miRNA-155 and Wnt5 a in lung tissues of rats in each group were detected by RT-PCR. The targeting relationship between miRNA155 and Wnt5 a was validated by a double luciferase reporter gene assay. Western Blot method was used to detect the expression levels of Wnt5 a, STAT3, and p-STAT3 in the lung tissue of rats in each group. The expression levels of IL-6 and TNF-α in lung homogenate were detected by ELISA. Results Compared with the normal control group, the pulmonary function indexes FVC, FEV1 and PEF in negative control group were significantly decreased, while FEV1/FVC was increased, and the expression level of miRNA-155 and Wnt5 a gene mRNA and protein in the negative control group increased significantly. Compared with the negative control group, the lung function of miRNA-155 expression inhibition group was improved in varying degrees, and the expression level of miRNA-155 and Wnt5 a gene mRNA and protein in the miRNA-155 expression inhibition group decreased significantly, the difference was statistically significant(P<0.05). The results of the double luciferase reporter gene assay showed that miRNA-155 and Wnt5 a could target binding. Compared with the normal control group(0.20±0.05) and(0.22±0.05), in the negative control group(1.35±0.05) and(1.11±0.05), and in the miRNA-155 expression inhibition group(1.01±0.06) and(0.52±0.07), the protein expression levels of STAT3 and p-STAT3 in lung tissue of rats were increased(P<0.05). The expression levels of STAT3 and p-STAT3 in the lung tissue of rats in miRNA-155 expression inhibition group were lower than those in the negative control group(P<0.05). Compared with the normal control group(95.92±8.78) pg/mL, and(88.99±8.12) pg/mL, the expression levels of IL-6 and TNF-α in lung tissue of negative control group(206.11±18.63) pg/mL and(177.54±13.12) pg/mL and those in miRNA-155 expression inhibition group(156.17±9.55) pg/mL and(131.94±9.25) pg/mL were increased(P<0.05). Compared with the negative control group, the expression levels of IL-6 and TNF-α in the lung tissue of rats in the miRNA-155 inhibited group were significantly decreased(P<0.05). Conclusion miRNA-155 activates the STAT3 signaling pathway by targeting Wnt5 a to promote the occurrence and development of bronchial asthma.
作者
梁音
张剑
王新卫
刘新年
张海英
LIANG Yin;ZHANG Jian;WANG Xin-wei;LIU Xin-nian;ZHANG Hai-ying(Department of Respiratory and Critical Medicine,Hubei No.3 People's Hospital of Jianghan University,Wuhan 430033,Hubei,China;Department of Clinical Laboratory9 Wuhan Jinyintan Hospital,Wuhan 430023,Hubei,China)
出处
《医学研究生学报》
CAS
北大核心
2020年第9期931-936,共6页
Journal of Medical Postgraduates
