Long non-coding RNA maternally expressed gene regulates cigarette smoke extract induced lung inflammation and human bronchial epithelial apoptosis via miR-149-3p

Abstract

Chronic obstructive pulmonary disease (COPD) has become a significant public health risk. Long non-coding RNAs (lncRNAs) have been identified as important factors involved in the proliferation, apoptosis and inflammatory cytokine expression of lung cells. Peripheral blood samples from 66 subjects (18 non-smokers, 24 smokers without COPD and 28 smokers with COPD) and HBE135-E6E7 cell treated with cigarette smoke extract (CSE) or not were used as the research object. The aim of the present study was to investigate the underlying mechanism of lncRNA maternally expressed gene 3 (MEG3) in COPD. Following transfection with microRNA (miR)-149-3p mimics, miR-negative control mimics, miR-149-3p inhibitor, miR-negative control inhibitor, small interfering (si)RNA targeting MEG3 (si-MEG3) and si-negative control (si-NC), levels of MEG3 and microRNA (miR)-149-3p were detected using reverse transcription-quantitative PCR, Proliferation and apoptosis were examined using the Cell Counting Kit-8 and flow cytometry assays, respectively. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Protein levels of B-cell lymphoma-2 (Bcl-2), cleaved-caspase-3, cleaved-caspase-9, phosphorylated (p)-p65, total (t)-p65, p-lkBα and t-lkBα were measured by western blotting. Luciferase assay was conducted to examine the relationship between MEG3 and miR-149-3p. LncRNA MEG3 was highly expressed, whereas miR-149-3p expression was downregulated in smokers with COPD peripheral blood samples, compared with non-smokers and smokers without COPD samples. Compared with untreated human bronchial epithelial (HBE) cells, MEG3 expression was increased in cigarette smoke extract (CSE)-treated HBE cells. Compared with CSE-treated HBE cells transfected with si-NC, MEG3 knockdown promoted cell proliferation and inhibited apoptosis in CSE-treated HBE cells transfected with si-MEG3, and it also decreased the levels of IL-6, TNF-α, Bcl-2 and increased cleaved-caspase-3 and cleaved-caspase-9 in CSE-treated HBE cells transfected with si-MEG3. The luciferase assay demonstrated that miR-149-3p has target sites for MEG3. MEG3 was demonstrated to regulate the NF-κB signaling pathway by sponging miR-149-3p in CSE-treated HBE cells. In conclusion, these findings suggested that MEG3 promoted proliferation and inhibited apoptosis by regulating the NF-κB signal pathway via miR-149-3p in CSE-treated HBE cells. These results provide an insight for further verification and understanding of the molecular basis of COPD.

Keywords: apoptosis; chronic obstructive pulmonary disease; inflammation; long non-coding RNA maternally expressed gene 3; microRNA-149-3p.

Figure 1

Relative mRNA expression levels of long non-coding RNA MEG3 and miR-149-3p in blood samples from patients with COPD and CSE-induced cells. (A and B) mRNA expression levels of MEG3 and miR-149-3p were measured by RT-qPCR in non-smokers, smokers and smokers with COPD. (C) Correlation analysis of MEG3 and miR-149-3p mRNA expression levels in smokers with COPD. (D and E) Relative mRNA expression levels of MEG3 and miR-149-3p were detected in untreated (Ctrl) and CSE-induced human bronchial epithelial cells using RT-qPCR. ^**P<0.01. MEG3, maternally expressed gene 3; RT-q, reverse-transcription quantitative; miR, microRNA; CSE, cigarette smoke extract; COPD, chronic obstructive pulmonary disease.

Figure 2

Effects of MEG3 knockdown on cell proliferation, apoptosis and inflammatory cytokine expression in CSE-induced HBE cells. Human bronchial epithelial cells were treated with control (untreated HBE cells), CSE, CSE + si-NC or CSE + si-MEG3. (A) mRNA expression of MEG3 in treated HBE cells was measured using reverse transcription-quantitative PCR. (B) Cell proliferation was examined by the Cell Counting Kit-8 assay. (C and D) The levels of IL-6 and TNF-α were detected using ELISA. (E) Apoptosis was analyzed by flow cytometry. (F) Protein expression levels of Bcl-2, caspase-3 and caspase-9 were determined by western blotting. ^**P<0.01. MEG3, maternally expressed gene 3; miR, microRNA; CSE, cigarette smoke extract; OD, optical density; PI, propidium iodide; NC, negative control; IL, interleukin; TNF, tumor necrosis factor; si, small interfering RNA.

Figure 3

Relationship between MEG3 and miR-149-3p. (A) The DIANNA online tool predicted the putative binding sites of MEG3 and miR-149-3p and MUT sequence of MEG3 is shown in red. (B) Luciferase activity was evaluated in HBE cells co-transfected with MEG3-WT or MEG3-MUT and miR-NC or miR-149-3p mimics. (C and E) The expression of MEG3 or miR-149-3p was examined in HBE cells transfected with empty vector and MEG3. (D and F) Levels of MEG3 or miR-149-3p were measured in HBE cells transfected with si-NC, si-MEG3#1, and si-MEG3#2. ^**P<0.01. MEG3, maternally expressed gene 3; miR, microRNA; CSE, cigarette smoke extract; HBE, human bronchial epithelial; NC, negative control; si, small interfering RNA; WT, wild-type; MUT, mutant.

Figure 4

MEG3 regulatory effects on miR-149-3p-mediated HBE cell proliferation, apoptosis and inflammatory cytokine expression. HBE cells were treated with control (untreated HBE cells), CSE, CSE + anti-miR-NC, CSE + miR-149-3p inhibitor, CSE + si-MEG3, CSE + si-MEG3 + anti-miR-NC or CSE + si-MEG3 + miR-149-3p inhibitor. (A) The expression of miR-149-3p was determined using reverse-transcription quantitative PCR in treated HBE cells. (B) Cell proliferation of treated HBE cells was determined using the Cell Counting Kit-8 assay. (C and D) Apoptosis analyzed by flow cytometry. (E) Protein levels of Bcl-2, caspase-3 and caspase-9 were measured by western blotting. (F) ELISA was used to measure the levels of IL-6 and (G) TNF-α. ^**P<0.01. MEG3, maternally expressed gene 3; miR, microRNA; CSE, cigarette smoke extract; OD, optical density; PI, propidium iodide; NC, negative control; IL, interleukin; TNF, tumor necrosis factor; si, small interfering RNA.

Figure 5

MEG3 regulates the NF-κB signaling pathway by targeting miR-149-3p. HBE cells were treated with control (untreated HBE cells), CSE, CSE + anti-miR-NC, CSE + miR-149-3p inhibitor, CSE + si-MEG3, CSE + si-MEG3 + anti-miR-NC or CSE + si-MEG3 + miR-149-3p inhibitor. The protein expression levels of p-p65, t-p65, p-IκBα, p-IκBα in treated cells were determined by western blotting. ^**P<0.01. MEG3, maternally expressed gene 3; miR, microRNA; CSE, cigarette smoke extract; NC, negative control; p, phosphorylated; t, total; si, small interfering RNA.