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

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或10.02 ± 1.15比3.06 ± 0.82), 差异均有统计学意义(P<0.05);而miR-206 inhibitor可显著抑制细胞增殖率, 促进细胞发生凋亡, 差异均有统计学意义(P<0.05);细胞周期分布结果显示, 与HASMCs组比较, miR-206 mimic组处于S期与G2/M期的细胞比例明显升高(P<0.05), 而miR-206 inhibitor组处于S期与G2/M期的细胞比例显著下降(P<0.05), miR-NC组差异无统计学意义(P>0.05);Western blot实验结果显示, 正常HASMCs或转染miR-NC的细胞相比, miR-206 mimic可显著上调细胞周期相关蛋白cyclin D1(0.43 ± 0.07或0.41 ± 0.02比0.63 ± 0.17)和cyclin B1(0.47 ± 0.13或0.50 ± 0.09比0.79 ± 0.31)的表达, 抑制PTEN(0.34 ± 0.10或0.29 ± 0.05比0.14 ± 0.02)与周期蛋白P21(0.34 ± 0.03或0.30 ± 0.05比0.11 ± 0.02)和凋亡相关蛋白Caspase-3的表达(0.29 ± 0.03或0.31 ± 0.05比0.15 ± 0.03), 差异均有统计学意义(P<0.05)。而miR-206 inhibitor可明显抑制细胞周期相关蛋白cyclin D1与cyclin B1的表达, 促进PTEN及周期蛋白P21、Caspase-3的表达(P<0.05)。结论在COPD中miR-206可靶向抑制气道平滑肌细胞中PTEN蛋白的表达并调控细胞周期的进展, 从而上调细胞的增殖能力, 抑制其凋亡。

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±0.51 vs.4.02±0.19),and miR-206 inhibitor could significantly inhibit the expression of miR-206 in cells(1.86±0.32),the difference was statistically significant(P<0.05);MTT and apoptosis experiments showed that miR-206 mimcs could significantly promote the proliferation rate of cells compared with normal HASMCs or miR-NC transfected cells(0.62±0.14 or 0.57±0.09 vs.0.83±0.05),inhibit cell apoptosis(9.13±1.71 or 10.02±1.15 vs.3.06±0.82),the differences were statistically significant(P<0.05),while miR-206 inhibitor could significantly inhibit cell proliferation and promote cell apoptosis(P<0.05)The results of cell cycle distribution showed that compared with HASMCs group,the proportion of cells in S phase and G2/M phase in miR-206 mimcs group increased significantly(P<0.05),while the proportion of cells in S phase and G2/M phase in miR-206 inhibitor group decreased significantly(P<0.05),and there was no significant difference in miR-NC group(P>0.05).The results of Western blot showed that compared with normal HASMCs or miR-NC transfected cells,miR-206 mimcs could significantly upregulate the expression of cyclin D1(0.43±0.07 or 0.41±0.02 vs.0.63±0.17),and cyclin B1(0.47±0.13 or 0.50±0.09 vs.0.79±0.31),and inhibit the expression of PTEN(0.34±0.10 or 0.29±0.05 vs.0.14±0.02),cyclin p21(0.34±0.03 or 0.30±0.05 vs.0.11±0.02),and apoptosis related protein caspase-3(0.29±0.03 or 0.31±0.05 vs.0.15±0.03),the differences were statistically significant(P<0.05).miR-206 inhibitor could significantly inhibit the expression of cyclin D1 and cyclin B1,and promote the expression of PTEN,cyclin p21 and caspase-3(P<0.05).Conclusions In COPD patients,miR-206 could targeted inhibit the expression of PTEN protein in airway smooth muscle cells and regulate the progress of cell cycle,so as to up regulate the proliferation of cells and inhibit their apoptosis.