PM_2.5 downregulates miR-194-3p and accelerates apoptosis in cigarette-inflamed bronchial epithelium by targeting death-associated protein kinase 1

Figures & data

Figure 1 PM_2.5 increased the apoptotic responses in cigarette-inflamed HBEpiCs.

Notes: HBEpiCs were treated with normal media, CSS or PM_2.5-CSS for 24 h. (A) Caspase 3/7 activity with mitochondrial membrane potential in living HBEpiCs. CSS induced a decrease in TMRM signal (red) and an increase in caspase 3/7 signal (green), and PM_2.5-CSS further decreased TMRM signal (red) and further activated caspase 3/7 signal (green). The scale bar was 100 μm. (B) Western blot analysis of apoptotic proteins. Phosphorylated AKT decreased in CSS-treated HBEpiCs and further decreased in PM_2.5-CSS-treated HBEpiCs; cleaved caspase 3 increased in CSS-treated HBEpiCs and further increased in PM_2.5-CSS-treated HBEpiCs; cleaved caspase 9 increased significantly in PM_2.5-CSS-treated HBEpiCs. (C and D) TUNEL analysis also revealed that PM_2.5 further increased the proportion of TUNEL-positive cells in CSS-treated HBEpiCs. The scale bar was 100 μm. n=4 per group ***p<0.001.
Abbreviations: PM_2.5, fine particulate matter; HBEpiCs, human bronchial epithelial cells; CSS, cigarette smoke solution; TMRM, tetramethylrhodamine, methyl ester reagent; TUNEL, transferase-mediated deoxyuridine triphosphate-biotin nick end labeling; pro, pro-form; cle, cleaved form; DAPI, diamidino-phenyl-indole.

Figure 2 The expression of miR-194-3p and DAPK1 in HBEpiCs.

Notes: HBEpiCs were treated with normal media, CSS or PM_2.5-CSS for 24 h. (A) Relative expression of miR-194-3p normalized against the U6 endogenous control. miR-194-3p decreased significantly in PM_2.5-CSS-treated HBEpiCs. (B) A potential binding site between miR-194-3p and DAPK1 mRNA 3′-UTR. (C) Relative expression of DAPK1 mRNA normalized against the β-actin endogenous control. Expression of DAPK1 mRNA had no statistical difference. (D) Western blot analysis of DAPK1. DAPK1 protein increased significantly in PM_2.5-CSS-treated HBEpiCs. n=3 per group *p<0.05 and ***p<0.001.
Abbreviations: DAPK1, death associated protein kinase 1; HBEpiCs, human bronchial epithelial cells; CSS, cigarette smoke solution; PM_2.5, fine particulate matter; 3′-UTR, 3′ untranslated region.

Figure 3 miR-194-3p directly targeted 3′-UTR of DAPK1 mRNA.

Notes: (A) Wild-type and mutant binding sites of miR-194-3p in the 3′-UTR of DAPK1. (B) Luciferase analysis. The results showed that miR-194-3p mimics decreased the fluorescence intensity in cells transfected with DAPK1-3′-UTR-wt but did not change the fluorescence intensity in cells transfected with DAPK1-3′-UTR-mut. (n=6) (C) HBEpiCs were treated with normal media for 24 h. Relative expression of miR-194-3p normalized against the U6 endogenous control and DAPK1 mRNA normalized against the β-actin endogenous control in normal HBEpiCs transfected with miR-194-3p mimics, inhibitors or scrambled controls. (D) Western blot analysis of DAPK1 and caspases. DAPK1 and cleaved caspase 3 were upregulated. Normal HBEpiCs transfected with miR-194-3p mimics. n=3 per group *p<0.05, **p<0.01 and ***p<0.001.
Abbreviations: 3′-UTR, 3′ untranslated region; DAPK1, death associated protein kinase 1; CSS, cigarette smoke solution; HBEpiCs, human bronchial epithelial cells; pro, pro-form; cle, cleaved form; wt, wild-type; mut, mutant.

Figure 4 Overexpression of miR-194-3p decreased DAPK1 and apoptosis in PM_2.5-CSS HBEpiCs.

Notes: HBEpiCs were treated with PM_2.5-CSS for 24 h. (A) Relative expression of miR-194-3p normalized against the U6 endogenous control and DAPK1 mRNA normalized against the β-actin endogenous control in PM_2.5-CSS-treated HBEpiCs transfected with miR-194-3p mimics, inhibitors or scrambled controls. (B) Western blot analysis of DAPK1 and caspases. DAPK1 and cleaved caspase 3 were downregulated in PM_2.5-CSS-treated HBEpiCs transfected with miR-194-3p mimics, while upregulated in PM_2.5-CSS cells transfected with miR-194-3p inhibitors. (C and D) TUNEL analysis. The proportion of TUNEL-positive cells decreased significantly in PM_2.5-CSS-treated HBEpiCs transfected with miR-194-3p mimics, while increased in PM_2.5-CSS cells transfected with miR-194-3p inhibitors. n=3 per group *p<0.05, **p<0.01 and ***p<0.001.
Abbreviations: DAPK1, death associated protein kinase 1; PM_2.5, fine particulate matter; CSS, cigarette smoke solution; HBEpiCs, human bronchial epithelial cells; TUNEL, transferase-mediated deoxyuridine triphosphate-biotin nick end labeling; pro, pro-form; cle, cleaved form.