ABSTRACT
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.