MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium

Abstract

We have shown that smoking impacts bronchial airway gene expression and that heterogeneity in this response associates with smoking-related disease risk. In this study, we sought to determine whether microRNAs (miRNAs) play a role in regulating the airway gene expression response to smoking. We examined whole-genome miRNA and mRNA expression in bronchial airway epithelium from current and never smokers (n = 20) and found 28 miRNAs to be differentially expressed (P < 0.05) with the majority being down-regulated in smokers. We further identified a number of mRNAs whose expression level is highly inversely correlated with miRNA expression in vivo. Many of these mRNAs contain potential binding sites for the differentially expressed miRNAs in their 3'-untranslated region (UTR) and are themselves affected by smoking. We found that either increasing or decreasing the levels of mir-218 (a miRNA that is strongly affected by smoking) in both primary bronchial epithelial cells and H1299 cells was sufficient to cause a corresponding decrease or increase in the expression of predicted mir-218 mRNA targets, respectively. Further, mir-218 expression is reduced in primary bronchial epithelium exposed to cigarette smoke condensate (CSC), and alteration of mir-218 levels in these cells diminishes the induction of the predicted mir-218 target MAFG in response to CSC. These data indicate that mir-218 levels modulate the airway epithelial gene expression response to cigarette smoke and support a role for miRNAs in regulating host response to environmental toxins.

Fig. 1.

Unsupervised hierarchical clustering of 28 differentially expressed microRNAs across bronchial epithelium of smokers and nonsmokers. Red indicates higher expression, green indicates lower expression. In general, most miRNAs that are altered by smoking show decreased expression in the airway epithelium of smokers. A small number of current smokers group with never smoker samples on the basis of expression of these miRNAs, suggesting heterogeneity in the response to smoking.

Fig. 2.

(A) qRT-PCR validation of 4 differentially expressed miRNAs, mir-218, mir-128b, mir-181d, and mir-500 in bronchial epithelial samples. Expression of these miRNAs changes in the same direction between smokers and nonsmokers both in microarray and in PCR analysis. (B) Expression of mir-218 in primary human bronchial epithelial cells that were untreated (n = 4), DMSO treated (n = 6), and cigarette smoke condensate treated (CSC, n = 5). Mir-218 expression was reduced in CSC-treated cells with respect to untreated (P = 0.008) cells. In both A and B, error bars indicate standard error, and P values were determined using Student's t test.

Fig. 3.

Relationship between the expression of miRNAs and their predicted mRNA targets in airway epithelium of current and never smokers. The distribution of Pearson correlation coefficients calculated between the miRNA and predicted target mRNA expression for 6 of the 28 differentially expressed miRNAs is shown in red. All 232 miRNAs and their predicted targets are in black. There is a significant increase in the degree of negative correlation between differentially expressed miRNAs and their targets relative to all miRNAs and their targets, which reflects the inhibitory effects of miRNAs on target gene expression. These 6 miRNAs have the highest number of predicted mRNA targets with significantly anticorrelated expression in vivo.

Fig. 4.

Expression of genes differentially expressed by increasing mir-218 levels in vitro (P < 0.05) that are predicted to be targets of mir-218. The orange blocks designate genes that are also anticorrelated with mir-218 expression in vivo (Pearson correlation, P < 0.1). Blue blocks indicate genes that are also differentially expressed in smokers (P < 0.1). Red indicates higher expression, green indicates lower expression.

Fig. 5.

Overexpressing mir-218 attenuates the induction of MAFG expression by cigarette smoke condensate in NHBE. CSC induces MAFG expression 1.54-fold (Scrambled + DMSO vs. Scrambled + CSC). However, when mir-218 is overexpressed, CSC induces MAFG expression only 1.11-fold (mir-218 + CSC vs. mir-218 + DMSO). The significantly reduced induction of MAFG by cigarette smoke condensate in the presence of high levels of mir-218 (2-way ANOVA interaction effect, P = 0.000003) suggests that modulation of mir-218 contributes to the CSC-dependent regulation of MAFG expression.