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

Abstract

Objective: To evaluate the effect of a single nucleotide polymorphism (rs2910164) in the miR-146a precursor on the expression level of miR-146a, cyclooxygenase-2 (COX2), and production of prostaglandin E2 (PGE2) in lung tissue harvested from smokers with chronic obstructive pulmonary disease, as well as the lung function and disease stages from the same patient population.

Methods and results: One-hundred and sixty-eight smokers with diagnosed chronic obstructive pulmonary disease were recruited. The patients were genotyped for rs2910164 polymorphism using Sanger sequencing, and their lung function/disease stages were evaluated following Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria. Meanwhile, messenger ribonucleic acid and protein expression levels of miR-146a and COX2 as well as PGE2 production were determined in 66 lung tissue samples collected in the patients who received surgical treatment. We confirmed that COX2 is a validated target of miR-146a in human fibroblast cells, and identified the differential expression patterns of miR-146a and COX2 in each rs2910164 genotype group. We observed a significant association between rs2910164 in miR-146a and the levels of either COX2 or PGE2 using real-time polymerase chain reaction and Western blot. Consistently, we were able to demonstrate that the rs2910164 single nucleotide polymorphism has a functional effect on the baseline lung function in the study population.

Conclusion: In the present study, the rs2910164 CC and GC genotype was found to be associated with an improved lung function and milder disease stages, at least partially, mediated by its ability to increase in COX2 expression and PGE2 production.

Keywords: COPD; COX-2; SNP; lung function; miR-146a.

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.