MiR-192-5p在支气管哮喘气道炎症中的作用

Role of miR-192-5p in airway inflammation in bronchial asthma

目的探讨微小RNA-192-50(miR-192-5p)在支气管哮喘(简称哮喘)患者血浆中的表达,及其对肿瘤坏死因子-α(TNF-α)诱导的人气道平滑肌细胞(ASMCs)增殖、炎性因子的表达以及细胞周期相关蛋白的影响。方法将本院收治的65例急性哮喘患者作为哮喘组,并选取同时期年龄相仿的65例健康人员作为健康对照组。用实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测哮喘患者血浆中miR-192-5p的表达水平。将ASMCs随机分为空白组(常规培养,不做任何处理)、模型组(20 ng·mL^(-1)TNF-α处理24 h)、转染对照组(转染模拟物阴性对照mimic-NC后,用20 ng·mL^(-1)TNF-α处理24 h)和转染组(转染miR-192-5p mimic后用20 ng·mL^(-1)TNF-α处理24 h)。用噻唑蓝(MTT)法检测ASMCs的增殖活力;用酶联免疫吸附(ELISA)法检测ASMCs细胞上清液中炎性因子白细胞介素-1β(IL-1β)和IL-6水平;用蛋白质印迹法检测细胞周期相关蛋白Cyclin D1和CDK6蛋白的水平。结果miR-192-5p在健康对照组和哮喘组血浆中的相对表达分别为1.01±0.09,0.44±0.05,与健康对照组相比,哮喘组血浆中miR-192-5p显著低表达(P<0.05)。空白组、模型组、转染对照组、转染组ASMCs上清液中IL-1β的水平分别为(132.58±17.41),(519.48±38.94),(509.41±20.34)和(204.14±28.71)pg·mL^(-1),IL-6的水平分别为(86.99±6.83),(639.21±56.25),(656.72±69.35)和(301.86±34.98)pg·mL^(-1),Cyclin D1蛋白的表达水平分别为0.36±0.02,0.95±0.08,0.97±0.09和0.48±0.03;CDK6蛋白的表达水平分别为0.11±0.01,1.04±0.09,1.01±0.08和0.16±0.02。模型组与空白组,转染对照组与转染组比较,差异均有统计学意义(均P<0.05)。结论miR-192-5p在支气管哮喘患者血浆中表达下调,过表达miR-192-5p抑制TNF-α诱导的ASMCs增殖、炎症和细胞周期相关蛋白。

Objective To investigate the expression of microRNA-192-50(miR-192-5p)in the plasma of patients with bronchial asthma(abbreviated as asthma),and its effects on proliferation,inflammatory factor levels and cell cycle-related proteins of human airway smooth muscle cells(ASMCs)induced by tumor necrosis factor-α(TNF-α).Methods Sixty-five patients with acute asthma were selected as asthma group,and 65 health examiners with similar ages during the same period were selected as the healthy control group.Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression level of miR-192-5p in the plasma of asthma patients.ASMCs were randomly divided into blank group(conventional culture without any treatment),model group(20 ng·mL^(-1) TNF-αtreatment for 24 h),transfection control group(after transfection of mimic negative control mimic-NC,treated with 20 ng·mL^(-1) TNF-αfor 24 h),transfection group(transfected with miR-192-5p mimic and treated with 20 ng·mL^(-1) TNF-αfor 24 h).MTT method was used to detect the proliferation activity of ASMCs;enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors interleubin-1β(IL-1β)and IL-6 in the supernatant of ASMCs cells;Western blotting was used to analyze the levels of Cyclin D1 and CDK6 proteins related to cell cycle.Results The relative expression of miR-192-5p in the plasma of healthy control group and asthma group were 1.01±0.09 and 0.44±0.05,respectively.Compared with the healthy control group,the expression of miR-192-5p in the plasma of the asthma group was significantly lower(P<0.05).The levels of IL-1βin the ASMCs supernatants of blank group,model group,transfection control group,and transfection group were(132.58±17.41),(519.48±38.94),(509.41±20.34)and(204.14±28.71)pg·mL^(-1),the level of IL-6 were(86.99±6.83),(639.21±56.25),(656.72±69.35)and(301.86±34.98)pg·mL^(-1),the expression levels of Cyclin D1 protein were 0.36±0.02,0.95±0.08,0.97±0.09 and 0.48±0.03;the expression levels of CDK6 protein were 0.11±0.01,1.04±0.09,1.01±0.08 and 0.16±0.02,respectively.Compared with model group and blank group,transfection control group and transfection group,the differences were statistically significant(all P<0.05).Conclusion The expression of miR-192-5p is down-regulated in the plasma of patients with bronchial asthma.Overexpression of miR-192-5p inhibits the proliferation,inflammation and cell cycle-related proteins of ASMCs induced by TNF-α.