MicroRNA-221-3p alleviates cell apoptosis and inflammatory response by targeting cyclin dependent kinase inhibitor 1B in chronic obstructive pulmonary disease

Figures & data

Figure 1. MiR-221-3p exhibits a low expression in COPD tissues and 16HBE cells. (a) MiR-221-3p levels in the tissues of nonsmokers (n = 11), smokers without COPD (n = 16) and smokers with COPD (n = 21) were detected by RT-qPCR. (b) The viability of 16HBE cells in response to different concentrations (0%, 1%, 2%, 3%) of CSE for 24 h (the left panel) or in response to 2% CSE for 0 h, 12 h, 24 h, 36 h, 48 h (the right panel) was detected by CCK-8. (c) MiR-221-3p levels in 16HBE cells by treatment of different concentrations (0%, 1%, 2%, 3%) of CSE for 24 h (the left panel) or by 2% CSE for 0 h, 12 h, 24 h, 36 h, 48 h (the right panel) was detected by RT-qPCR. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 2. Overexpressed miR-221-3p suppressed the apoptosis and inflammation of 16HBE cells. (a) MiR-221-3p expression in the four groups. (b) The apoptosis of 16HBE cells in different groups. (c) The protein levels of Bax, cleaved caspase-3 and Bcl-2 in 16HBE cells from different groups. (d-e) The concentrations of inflammatory cytokines COX-2, IL-6, TNF-α and IL-1β in the four groups. **P < 0.01, ***P < 0.001

Figure 3. Overexpressed miR-221-3p suppressed the apoptosis and inflammation of 16HBE cells. (a) MiR-221-3p silencing efficiency was detected by RT-qPCR. (b) The apoptosis of 16HBE cells under the condition of miR-221-3p deficiency was detected by CCK-8. (c) The protein levels of Bax, cleaved caspase-3 and Bcl-2 in 16HBE cells under the condition of miR-221-3p deficiency were detected by western blotting. (d) The concentrations of inflammatory cytokines COX-2, IL-6, TNF-α and IL-1β under the condition of miR-221-3p deficiency were detected by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 4. CDKN1B was targeted by miR-221-3p. (a) The expressions of the 5 mRNAs that shared binding site with miR-221-3p in 16HBE cells with or without CSE stimulation. (b) CDKN1B expression in the tissues of nonsmokers (n = 11), smokers without COPD (n = 16) and smokers with COPD (n = 21). (c) The binding site of CDKN1B 3ʹUTR with miR-221-3p. (d) The binding capability of CDKN1B with miR-221-3p was assessed by luciferase reporter assay. (e) The correlation of CDKN1B level and miR-221-3p level in 21 COPD tissues was determined using Pearson correlation analysis. (f) Effects of miR-221-3p on CDKN1B mRNA and protein levels were detected by RT-qPCR and western blotting. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 5. CDKN1B overexpression counteracted the influence of miR-221-3p on the apoptosis and inflammation of 16HBE cells. (a) The overexpression efficiency of CDKN1B was detected by western blotting. (b) 16HBE cell apoptosis with transfection of indicated plasmids was detected by annexin V-FITC/PI staining and analyzed by flow cytometry. (c) The protein levels of Bax, Bcl-2 and Cleaved Caspase-3 in 16HBE cells from different groups were detected by western blotting. (d-e) The concentrations of inflammatory cytokines COX-2, IL-6, TNF-α and IL-1β in the four groups were detected by ELISA. **P < 0.01, ***P < 0.001