CD147 Promoted Epithelial Mesenchymal Transition in Airway Epithelial Cells Induced by Cigarette Smoke via Oxidative Stress Signaling Pathway

Figures & data

Table 1. The primers for real-time PCR.

Gene	Sequence
human β-actin	F: TGGCACCCAGCACAATGAA
	R: CTAAGTCATAGTCCGCCTAGAAGCA
human CD147	F: ATCCAGTGGTGGTTTGAAGGG
	R: ACGAGCGTGTCGATGGAGAT
human Ecadherin	F: AAAGGCCCATTTCCTAAAAACCT
	R: TGCGTTCTCTATCCAGAGGCT
human α-SMA	F: CCTGTGTTGTGGTTTACACTGG
	R: GGGGGAATTATCTTTCCTGGTCC
human ZO1	F: CTGGTGAAATCCCGGAAAAATGA
	R: TTGCTGCCAAACTATCTTGTGA
human Vimentin	F: TGCCGTTGAAGCTGCTAACTA
	R: CCAGAGGGAGTGAATCCAGATTA
human HO1	F: ATGGCCTCCCTGTACCACATC
	R: TGTTGCGCTCAATCTCCTCCT
human GSTP	F: CATCTACACCAACTATGAGGCG
	R: AGCAGGGTCTCAAAAGGCTTC
human NQO1	F: GAAGAGCACTGATCGTACTGGC
	R: GGATACTGAAAGTTCGCAGGG
human GCLC	F: GGAGACCAGAGTATGGGAGTT
	R: CCGGCGTTTTCGCATGTTG
mouse β-actin	F: AGAGGGAAATCGTGCGTGAC
	R: CAATAGTGATGACCTGGCCGT
mouse CD147	F: CCTGCATACGAAGTACATAGTGG
	R: TGATTTCTTTCCGACCTTGATCC
mouse Ecadherin	F: CAGTTCCGAGGTCTACACCTT
	R: TGAATCGGGAGTCTTCCGAAAA
mouse α-SMA	F: GGCACCACTGAACCCTAAGG
	R: ACAATACCAGTTGTACGTCCAGA
mouse ZO1	F: GAGCGGGCTACCTTACTGAAC
	R: GTCATCTCTTTCCGAGGCATTAG
mouse Vimentin	F: GCTGCGAGAGAAATTGCAGGA
	R: CCACTTTCCGTTCAAGGTCAAG
mouse HO1	F: AAGCCGAGAATGCTGAGTTCA
	R: GCCGTGTAGATATGGTACAAGGA
mouse GSTP	F: ATGCCACCATACACCATTGTC
	R: GGGAGCTGCCCATACAGAC
mouse NQO1	F: AGAGAGTGCTCGTAGCAGGAT
	R: GTGGTGATAGAAAGCAAGGTCTT
mouse GCLC	F: CTACCACGCAGTCAAGGACC
	R: CCTCCATTCAGTAACAACTGGAC

Figure 1. Cigarette smoke induced CD147 expression in murine lung tissue and HBE cells. (a, b) CD147 mRNA (a) and protein (b) expression in murine lung tissue; (c, d) CD147 mRNA (c) and protein (d) expression in HBE cells treated by CSE ranged from 0–10%; (e, f) CD147 mRNA (e) and protein (f) expression in HBE cells treated by 10% CSE for 0–48 h. (animal study was performed with six replicates in each group, and cell experiments were performed in triplicates, *p < 0.05; **p < 0.01; ***p < 0.001, compared with CON or 0).

Figure 2. Cigarette smoke upregulated α-SMA, Vimentin expression and downregulated Ecadherin, ZO1 expression both in vivo and in vitro. (a–d) Ecadherin (a), α-SMA (b), ZO1 (c) and Vimentin (d) mRNA expression in murine lung tissue; (e) representative pictures of Ecadherin (left panel), α-SMA (middle left panel), ZO1 (middle right panel) and Vimentin (right panel) protein expression in murine lung tissue by IHC (scale bar, 50 μm); (f–i) Ecadherin (f), α-SMA (g), ZO1 (h) and Vimentin (i) mRNA expression in HBE cells treated by CSE in a series of concentrations; (j–m) Ecadherin (j), α-SMA (k), ZO1 (l) and Vimentin (m) mRNA expression in HBE cells treated by 10% CSE for 0–48 h; (n, o) Ecadherin, α-SMA, ZO1 and Vimentin protein expression in HBE cells treated by CSE in a series of concentrations (n) or 10% CSE for 0–48 h (o). (Animal study was performed with six replicates in each group, and cell experiments were performed in triplicates, *p < 0.05; **p < 0.01; ***p < 0.001, compared with CON or 0).

Figure 3. The effect of CD147 knockdown on CSE induced EMT markers expression in HBE cells. (a–d) Ecadherin (a), α-SMA (b), ZO1 (c) and Vimentin (d) mRNA expression in HBE cells transfected with control or CD147 siRNA and then treated by 10% CSE for 48 h; (e) Ecadherin, α-SMA, ZO1 and Vimentin protein expression in HBE cells transfected with control or CD147 siRNA and then treated by 10% CSE for 48 h; (f) representative images of Ecadherin (left panel), α-SMA (middle left panel), ZO1 (middle right panel) and Vimentin (right panel) protein expression in HBE cells by IF (All analyses were performed in triplicates, *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4. Cigarette smoke induced oxidative stress response in vivo and in vitro. (a) representative pictures of 8-OHdG expression in murine lung tissue by IHC (scale bar, 50 μm); (b) HO1, GSTP, NQO1 and GCLC mRNA expression in murine lung tissue; (c) relative H₂O₂ production in culture medium in HBE cells treated by 10% CSE for 24 h; (d) mean flurescence intensity (MFI) of intracellular ROS treated by 10% CSE for 24 h; (e–h) HO1 (e), GSTP (f), NQO1 (g) and GCLC (h) mRNA expression in HBE cells treated by 10% CSE (animal study was performed with six replicates in each group, -and cell experiments were performed in triplicates, *p < 0.05; **p < 0.01; ***p < 0.001, compared with CON or 0).

Figure 5. Oxidative stress was involved in cigarette smoke induced EMT process. (a, b) the effect of NAC (a) or H₂O₂ (b) on Ecadherin mRNA expression in HBE cells treated by 10% CSE for 48 h; (c, d) the effect of NAC (c) or H₂O₂ (d) on α-SMA mRNA expression in HBE cells treated by 10% CSE for 48 h; (e, f) The effect of NAC (e) or H₂O₂ (f) on ZO1 mRNA expression in HBE cells treated by 10% CSE for 48 h; (g, h) the effect of NAC (g) or H₂O₂ (h) on Vimentin mRNA expression in HBE cells treated by 10% CSE for 48 h; (i, j) the effect of NAC (i) or H₂O₂ (j) on Ecadherin, α-SMA, ZO1 and Vimentin protein expression in HBE cells treated by 10% CSE for 48 h. (All analyses were performed in triplicates, *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 6. Cigarette smoke promoted EMT in HBE cells via oxidative stress signal pathway. (a) Relative H₂O₂ production in culture medium in HBE cells transfected with control or CD147 siRNA and then treated by 10% CSE for 24 h; (b) mean flurescence intensity (MFI) of intracellular ROS in HBE cells transfected with control or CD147 siRNA and then treated by 10% CSE for 24 h; (c–f) HO1 (c), GSTP (d), NQO1 (e) and GCLC (f) mRNA expression in HBE cells transfected with control or CD147 siRNA and then treated by 10% CSE for 24 h; (g–j) Ecadherin (g), α-SMA (h), ZO1 (i) and Vimentin (j) mRNA expression in HBE cells transfected with control or CD147 siRNA and then treated by 10% CSE for 48 h with or without preincubation of 0.5 mM H₂O₂; (k) Ecadherin, α-SMA, ZO1 and Vimentin protein expression in HBE cells transfected with control or CD147 siRNA and then treated by 10% CSE for 48 h with or without preincubation of 0.5 mM H₂O₂. (l) Representative IF images of Ecadherin (left panel), α-SMA (middle left panel), ZO1 (middle right panel) and Vimentin (right panel) protein expression in HBE cells transfected with control or CD147 siRNA and then treated by 10% CSE for 48 h with or without preincubation of 0.5 mM H₂O₂. (All analyses were performed in four replicates, *p < 0.05; **p < 0.01; ***p < 0.001).