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Long non-coding maternally expressed gene 3 regulates cigarette smoke extract-induced apoptosis, inflammation and cytotoxicity by sponging miR-181a-2-3p in 16HBE cells.

ABSTRACT: Accumulating evidence has suggested that long non-coding (lnc)RNAs are widely involved in the progression of multiple diseases, including chronic obstructive pulmonary disease (COPD). The aim of the present study was to explore the function and molecule mechanism of maternally expressed gene 3 (MEG3) in cigarette smoke extract (CSE)-treated 16HBE cells. Cell viability and apoptosis were evaluated using Cell Counting Kit-8 analysis and flow cytometry, respectively. Western blot analysis was carried out to determine the protein levels of Bcl-2, Bax and cleaved caspase-3. ELISA assays were utilized to measure the protein levels of IL-1? and IL-6 and TNF-?. Cytotoxicity was assessed using a lactate dehydrogenase release assay. The expression levels of MEG3 and microRNA (miR)-181a-2-3p were detected using reverse transcription-quantitative PCR. The interaction between miR-181a-2-3p and MEG3 was predicted using DIANA tools and verified by a dual-luciferase reporter assay and RNA Immunoprecipitation assay. MEG3 expression was enhanced while miR-181a-2-3p abundance was reduced in the serum of patients with COPD and CSE-treated 16HBE cells. MEG3-knockdown or miR-181a-2-3p-overexpression inhibited CSE-induced apoptosis, inflammation and cytotoxicity in 16HBE cells. Moreover, miR-181a-2-3p directly bind to MEG3 and its knockdown reversed the inhibitory effect of MEG3 interference on apoptosis, inflammation and cytotoxicity in CSE-treated 16HBE cells. Overall, MEG3-knockdown suppressed CSE-induced apoptosis, inflammation and cytotoxicity in 16HBE cells by upregulating miR-181a-2-3p, providing a promising therapeutic target for treatment of CSE-induced COPD.

SUBMITTER: Fan S

PROVIDER: S-EPMC7693283 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Long non-coding maternally expressed gene 3 regulates cigarette smoke extract-induced apoptosis, inflammation and cytotoxicity by sponging miR-181a-2-3p in 16HBE cells.

Fan Shiming S Ren Yan Y Zhang Wenli W Zhang Huawei H Wang Cheng C

Oncology letters 20201117 1

Accumulating evidence has suggested that long non-coding (lnc)RNAs are widely involved in the progression of multiple diseases, including chronic obstructive pulmonary disease (COPD). The aim of the present study was to explore the function and molecule mechanism of maternally expressed gene 3 (MEG3) in cigarette smoke extract (CSE)-treated 16HBE cells. Cell viability and apoptosis were evaluated using Cell Counting Kit-8 analysis and flow cytometry, respectively. Western blot analysis was carri ...[more]

PMID: 33262837

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Project description: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.

Dataset Information

Long non-coding maternally expressed gene 3 regulates cigarette smoke extract-induced apoptosis, inflammation and cytotoxicity by sponging miR-181a-2-3p in 16HBE cells.

Publications

Long non-coding maternally expressed gene 3 regulates cigarette smoke extract-induced apoptosis, inflammation and cytotoxicity by sponging miR-181a-2-3p in 16HBE cells.

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