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. 2024 May 25:19:1141-1151.
doi: 10.2147/COPD.S453593. eCollection 2024.

A miRNA-21-Mediated PTEN/Akt/NF-κB Axis Promotes Chronic Obstructive Pulmonary Disease Pathogenesis

Affiliations

A miRNA-21-Mediated PTEN/Akt/NF-κB Axis Promotes Chronic Obstructive Pulmonary Disease Pathogenesis

Xiaoyan Sai et al. Int J Chron Obstruct Pulmon Dis. .

Abstract

Background: This study sought to explore the underlying mechanism of miR-21 mediated apoptosis and inflammation in chronic obstructive pulmonary disease (COPD) induced by cigarette smoke (CS).

Methods: We detected levels and PTEN/Akt/NF-κB axis protein levels in peripheral lung tissues of COPD patients and CS-exposed mice and HBE cells. Western blotting assay was used to determine the expression of cleaved caspase-3. IL-6 and IL-8 protein was detected in cell supernatant from cells by ELISA. HBE cells were transfected with a miR-21 inhibitor, and co-culture with A549.

Results: Increased miR-21 expression, reduced PTEN expression and following activation of Akt in in peripheral lung tissues of COPD patients and CS-exposed mice and HBE cells. Inhibition of miR-21 showed enhanced PTEN levels and reduced the expression of phosphorylated form of Akt and NF-κB. Decreased expression of cleaved caspase-3, IL-6 and IL-8 in A549 cells co cultured with HBE cells transfected with miR-21 inhibitor compared with transfected with miR-21 control inhibitor.

Conclusion: MiR-21 contributes to COPD pathogenesis by modulating apoptosis and inflammation through the PTEN/Akt/NF-κB pathway. Targeting miR-21 may increase PTEN expression and inhibit Akt/NF-κB pathway, offering potential diagnostic and therapeutic value in COPD management.

Keywords: COPD; PTEN/Akt/NF-κB axis; apoptosis; inflammation; miR-21.

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Conflict of interest statement

Xiaoyan Sai, Chu Qin, and Zixiao Zhang are co-first authors for this study. The authors declare that there is no conflict of interest in this work.

Figures

Figure 1
Figure 1
Enhanced miR-21 expression in lung tissue of COPD patients. Assessment of MiR-21 via qRT-PCR across groups: Con-NS (non-smokers, no COPD) (n =5), Con-S (smokers, no COPD) (n =7), and COPD (patients with COPD) (n =7). Data represent mean ± standard deviation. Statistical thresholds: *P < 0.05, (via one-way ANOVA).
Figure 2
Figure 2
Decreased PTEN expression, activating Akt/NF-κB pathway, and increasing inflammation and apoptosis in COPD patients. (A) Protein levels of PTEN, phosphorylated Akt (p-Akt), Akt, phosphorylated NF-κB (p-NF-κB), NF-κB, and β-actin in Western blot analysis. (B) Comparison of PTEN, p-Akt, p-NF-κB protein levels. (C) IL-6 and (D) IL-8 levels were measured by ELISA. (E)Western blot were determined and (F) relative protein levels of cleaved caspase-3 in the peripheral lung tissue. Density of bands measured using ImageJ. β-actin for normalization. Group classification: Con-NS (non-smokers, no COPD) (n =5), Con-S (smokers, no COPD) (n =7), COPD (COPD patients) (n =7). Data: average values ± standard deviation. Statistical thresholds: *P < 0.05, **P < 0.01, via one-way ANOVA.
Figure 3
Figure 3
Increased miR-21 expression, reduced PTEN expression and following activation of Akt in lung from COPD mice. (A) Lung tissue of mice exposed to air and cigarette smoke (CS) analyzed via Hematoxylin-eosin staining, revealing tissue destruction and airway dilation (original magnification ×100). Bar: 200 μm. (B and C) Evaluation of lung architecture through Mean Linear Intercept (MLI) quantification and Emphysema assessment via Mean Alveolar Numbers (MAN). (D) Determination of miR-21 levels in lung tissue by quantitative Real-Time PCR (qRT-PCR). (E) Protein expression of PTEN, phosphorylated Akt (p-Akt), Akt, phosphorylated NF-κB (p-NF-κB), NF-κB assessed by Western Blot. (F) Band density quantified using ImageJ. Data presented as mean ± standard deviation, n = 3. Statistical analysis: *P < 0.05, **P < 0.01, via one-way ANOVA.
Figure 4
Figure 4
CSE inducing increase of miR-21 and decrease of PTEN in HBE cells. (A) Exposure of HBE cells to 5% CSE at intervals of 12h, 24h, and 48h. (B) Assessment of miR-21 levels via quantitative Real-Time PCR (qRT-PCR). (C) Protein expression evaluated using Western blot, with relative levels determined. Band densities quantified by ImageJ. Data expressed as mean ± standard deviation, n = 3. Statistical significance established at *P < 0.05, **P < 0.01, through one-way ANOVA.
Figure 5
Figure 5
miR-21 targeting PTEN. (A) The binding site of miR-21 and PTEN; (B) Luciferase reporter assay was used to detect the regulatory association between miR-21 and PTEN. Data expressed as mean ± standard deviation, n = 3. Statistical significance established at *P < 0.05.
Figure 6
Figure 6
MiR-21 activating Akt/NF-κB pathway via inhibition of PTEN in HBE cells. (A) Treatment of HBE cells involved miR-21 inhibitor and a control inhibitor over a 48-hour period, followed by CSE exposure for another 48 hours. (B) MiR-21 expression quantified using qRT-PCR. Analysis of PTEN, p-Akt, and p-NF-κB protein levels via Western blot. (C) ImageJ utilized for measuring band densities. Labels: MiR-21i (miR-21 inhibitor), Con-inhibitor (control for miR-21 inhibitor). Statistical data: averages ± standard deviation, n = 3, significance denoted as *P < 0.05, **P < 0.01, determined through one-way ANOVA.
Figure 7
Figure 7
Decreased inflammation and apoptosis of A549 in co culture with HBE cells transfected with miR-21 inhibitor. HBE cells were co-cultured with A549 cells which were transfected with control inhibitor or miR-21 inhibitor and then treated with CSE for 48h. A549 cells are divided into four groups according to co-cultured HBE cells: control, CSE, CSE plus control inhibitor and CSE plus miR-21 inhibitor. (A) IL-6 and (B) IL-8 levels were measured by ELISA. (C) TUNEL-positive staining (in green) and DAPI (in blue) of A549 cells co-cultured with HBE cells (200x). (D) Quantitative analysis of TUNEL positive cells. (E)Western blot were determined and (F) relative protein levels of caspase-3 in A549 cells co-cultured with HBE cells. (G) Cell viability was measured by CCK-8 assay after co-cultured with HBE cells. Dates were mean ± SD, n=3, *P<0.05; **P < 0.01; ***P < 0.001.

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