Effect of a single nucleotide polymorphism in miR-146a on COX-2 protein expression and lung function in smokers with chronic obstructive pulmonary disease

Figures & data

Table 1 Demographic data and clinical characteristics of the participants stratified by rs2910164 genotype

Genotype	GG (n=72)	GC (n=73)	CC (n=23)	P-value
Age, mean ± SD	61.2±4.3	61.4±5.4	60.8±4.6	0.872
Sex (M/F)	49/23	46/27	16/7	0.757
Height (cm), mean ± SD	170.2±8.6	171.1±8.4	170.3±9.6	0.809
Weight (kg), mean ± SD	68.4±7.4	69.3±6.4	69.6±7.1	0.671
Smoking (pack years), mean ± SD	44.1±6.3	44.4±6.8	43.1±5.8	0.702
Smoking status	72	73	23	0.729
Current smoker	41	37	13
Ex-smoker	31	36	10
GOLD stage				<0.01
I	3	4	2
II	19	37	13
III	37	25	6
IV	13	7	2
FVC (% of predicted value), mean ± SD	88.3±6.9	91.3±7.1	92.3±4.2	<0.01
FEV1 (% of predicted value), mean ± SD	46.7±3.3	53.7±4.6	54.3±3.2	<0.01
DLCO (% of predicted value), mean ± SD	60.1±5.4	65.2±5.7	66.4±4.2	<0.01

Abbreviations: GOLD, Global Initiative for Chronic Obstructive Lung Disease; FVC, forced vital capacity; FEV₁, forced expiratory volume in 1 second; DLCO, diffusion capacity for carbon monoxide; M, male; F, female.

Figure 1 The effect of rs2910164 polymorphism on PGE2 concentration in lung tissues.

Notes: (A) Expression level of miR-146a (lung tissues) in each genotype group; (B) mRNA expression level of COX2 (lung tissues) in each genotype group; (C) protein expression level of COX2 (lung tissues) in each genotype group; (D) relative density of Western blot in C by densitometry analysis. (E) PGE2 production level (lung tissues) in each genotype group.
Abbreviations: mRNA, messenger ribonucleic acid; PGE2, prostaglandin E2; COX2, cyclooxygenase-2.

Figure 2 Luciferase assay with wild-type and mutant COX2 3’UTR.

Notes: (A) Schematic comparison of the “seed sequence” in 3′ UTR of COX2 and has-miR-146a; (B) has-miR-146a could suppress the luciferase activity of WT1 COX2 3′UTR, but not mutant COX2 3′UTR.

Abbreviations: COX2, cyclooxygenase-2; UTR, untranslated region; Luc, luciferase.

Figure 3 Comparison of the expression of COX2 (lung tissues) in each genotype group by immunohistochemistry.

Notes: (A) GG; (B) GC; (C) CC.
Abbreviation: COX2, cyclooxygenase-2.

Figure 4 Introduction of miR-146a mimics downregulated the expression of COX2.

Notes: (A) Transfection of miR-146a significantly increased the expression level of miR-146 in fibroblast cells. (B) Transfection of miR-146a mimics significantly suppressed the mRNA expression level of COX2 in human fibroblast cells; (C) transfection of miR-146a mimics significantly suppressed the protein expression level of COX2 in human fibroblast cells; (D) densitometrically analyzed Western blot results of knockdown of COX2 by miR-146a mimics.

Abbreviations: COX2, cyclooxygenase-2; mRNA, messenger ribonucleic acid; h, hour(s).

Figure 5 Introduction of miR-146a inhibitors upregulated the expression of COX2.

Notes: (A) Transfection of miR-146a significantly decreased the expression level of miR-146 in fibroblast cells. (B) Transfection of miR-146a inhibitors significantly promoted the mRNA expression level of COX2 in human fibroblast cells; (C) transfection of miR-146a inhibitors significantly promoted the protein expression level of COX2 in human fibroblast cells; (D) densitometrically analyzed Western blot results of knockdown of COX2 by miR-146a inhibitors.

Abbreviations: COX2, cyclooxygenase-2; mRNA, messenger ribonucleic acid; h, hour(s).

Figure 6 Introduction of miR-146a mimics and inhibitors on the expression of COXs in pulmonary artery smooth muscle cells.

Notes: (A) Transfection of miR-146a mimics significantly suppressed the mRNA expression level of COX2 in human pulmonary smooth muscle cells; (B) transfection of miR-146a mimics significantly suppressed the protein expression level of COX2 in human pulmonary smooth muscle cells; (C) densitometrically analyzed Western blot results of knockdown of COX2 by miR-146a mimics. (D) Transfection of miR-146a inhibitors significantly promoted the mRNA expression level of COX2 in human pulmonary smooth muscle cells; (E) transfection of miR-146a inhibitors significantly promoted the protein expression level of COX2 in human pulmonary smooth muscle cells; (F) densitometrically analyzed Western blot results of knockdown of COX2 by miR-146a inhibitors.
Abbreviations: COX2, cyclooxygenase-2; mRNA, messenger ribonucleic acid.