微小RNA-206在慢性阻塞性肺疾病的表达及其对气道平滑肌细胞增殖的影响  

Study on the mechanism of the expression of microRNA-206 in chronic obstructive pulmonary disease and its effect on the proliferation of human airway smooth muscle cells

作　　者：陶柯衡 韩育宁[1] 何进喜[1] Tao Keheng;Han Yuning;He Jinxi(Department of General Thoracic Surgery,General Hospital of Ningxia Medical University,Yinchuan 750000,China)

机构地区：[1]宁夏医科大学总医院普胸外科,银川750000

出　　处：《中国医师进修杂志》2022年第2期101-108,共8页Chinese Journal of Postgraduates of Medicine

基　　金：宁夏回族自治区重点研发项目(2019BEG03012)。

摘　　要：目的探究微小RNA(miR)-206在慢性阻塞性肺疾病(COPD)中的表达及其对气道平滑肌细胞增殖的影响及相关作用机制。方法收集2017年9月至2018年9月宁夏医科大学总医院行肺部减容手术的15例COPD患者的肺组织样本(COPD组)和15例无COPD病史的肺部良性肿瘤患者的对照肺组织样本, 利用基因芯片技术分别对4例COPD患者和正常对照肺组织进行miRNA及mRNA组学分析, 并使用逆转录聚合酶链反应(RT-PCR)进行验证在COPD患者和健康对照肺组织中差异表达的miRNA;生物信息学预测, 双荧光素酶基因报告实验检测miR-206在人气道平滑肌细胞(HASMCs)的作用靶基因;将miR-206 mimic或miR-206 inhibitor瞬时转染入HASMCs中, RT-PCR检测转染后的miR-206表达水平;噻唑蓝(MTT)实验、流式细胞周期及细胞凋亡实验检测miR-206对HASMCs的增殖、细胞周期及凋亡的影响;Western blot实验检测miR-206对HASMCs中PTEN与细胞周期和凋亡蛋白相关蛋白的表达影响。结果 miRNA及mRNA组学分析结果显示, 与对照肺组织样本相比, COPD组中miR-206、miR-3187-5p、miR-124表达显著上调(0.09 ± 0.01比2.17 ± 0.57、0.60 ± 0.04比1.32 ± 0.15、0.22 ± 0.08比1.09 ± 0.23)(P<0.05), 而miR-574、miR-337-3p表达明显下降(0.79 ± 0.03比0.15 ± 0.02、0.95 ± 0.02比0.17 ± 0.01)(P<0.05)。利用RT-PCR在15例COPD肺组织检测5种miRNA的表达, 结果显示其表达与微阵列中的表达情况一致;miRNA靶基因预测结果显示, miR-206能靶向抑制PTEN的表达;在HASMCs中转染后, RT-PCR结果显示, 与miR-健康对照组(NC)(4.02 ± 0.19)相比, miR-206 mimic可明显上调细胞中miR-206的表达水平(7.44 ± 0.51), 而miR-206 inhibitor可显著抑制细胞中miR-206的表达(1.86 ± 0.32), 差异均有统计学意义(P<0.05);MTT与凋亡实验结果显示, 与正常HASMCs或转染miR-NC的细胞相比, miR-206 mimic可显著促进细胞的增殖率(0.62 ± 0.14或0.57 ± 0.09比0.83 ± 0.05), 抑制细胞的凋亡(9.13 ± 1.71�Objective To investigate the expression of microRNA(miR)-206 in chronic obstructive pulmonary disease(COPD)and its effect on the proliferation of human airway smooth muscle cells(HASMCs)and to explore its mechanism.Methods Lung tissue samples of 15 patients with COPD(COPD group)who underwent lung volume reduction surgery in the General Hospital of Ningxia Medical University from September 2017 to September 2018 and of 15 patients with benign lung tumors without a history of COPD were collected.Microarray technology was used to analyze the miR and RNA omics in lung tissues of 4 COPD patients and normal controls,and reverse transcriptase polymerase chain reaction(RT-PCR)was used to verify the results.Bioinformatics and double luciferase gene reporting assay were used to detect the target genes of miR-206 in HASMCs.The miR-206 mimic/inhibitor was transfected into HASMCs by liposome transfection technology,and the expression level of miR-206 was detected by RT-PCR.Methyl thiazolyl tetrazolium(MTT),flow cytometry and apoptosis assay were used to detect the effects of miR-206 on the proliferation,cell cycle and apoptosis of HASMCs.The expression of PTEN,cell cycle and apoptotic protein in HASMCs was detected by Western blot.Results The results of miR and mRNA omics analysis showed that the expressions of miR-206,miR-3187-5p and miR-124 in COPD group were significantly up-regulated(0.09±0.01 vs.2.17±0.57,0.60±0.04 vs.1.32±0.15,0.22±0.08 vs.1.09±0.23)(P<0.05),while the expressions of miR-574 and miR-337-3p decreased significantly(0.79±0.03 vs.0.15±0.02,0.95±0.02 vs.0.17±0.01)(P<0.05).RT-PCR was used to detect the expression of these five miRNAs in 15 COPD lung tissues,and the results showed that their expression was consistent with that in microarray.The prediction results of miRNA target genes showed that miR-206 could directly inhibit the expression of PTEN.RT-PCR results showed that the expression of miR-206 in miR-206 transfected HASMCs was significantly higher than that in miR-NC transfected group(7.44±0.

关 键 词：肺疾病 慢性阻塞性 肌细胞 平滑肌 核糖核酸酶类 细胞周期 

分 类 号：R563.9[医药卫生—呼吸系统]