Current Smoking is Associated with Decreased Expression of miR-335-5p in Parenchymal Lung Fibroblasts.

Jennie Ong,Ilse Boudewijn,Brian Oliver,Joost Kluiver,Corry-Anke Brandsma,Klaas Kok,Alen Faiz,Anke Van Den Berg,Wim Timens,Maarten Van Den Berge,Cornelis Vermeulen,Martijn Terpstra

doi:10.3390/ijms20205176

Abstract

Cigarette smoking causes lung inflammation and tissue damage. Lung fibroblasts play a major role in tissue repair. Previous studies have reported smoking-associated changes in fibroblast responses and methylation patterns. Our aim was to identify the effect of current smoking on miRNA expression in primary lung fibroblasts. Small RNA sequencing was performed on lung fibroblasts from nine current and six ex-smokers with normal lung function. MiR-335-5p and miR-335-3p were significantly downregulated in lung fibroblasts from current compared to ex-smokers (false discovery rate (FDR) <0.05). Differential miR-335-5p expression was validated with RT-qPCR (p-value = 0.01). The results were validated in lung tissue from current and ex-smokers and in bronchial biopsies from non-diseased smokers and never-smokers (p-value <0.05). The methylation pattern of the miR-335 host gene, determined by methylation-specific qPCR, did not differ between current and ex-smokers. To obtain insights into the genes regulated by miR-335-5p in fibroblasts, we overlapped all proven miR-335-5p targets with our previously published miRNA targetome data in lung fibroblasts. This revealed Rb1, CARF, and SGK3 as likely targets of miR-335-5p in lung fibroblasts. Our study indicates that miR-335-5p downregulation due to current smoking may affect its function in lung fibroblasts by targeting Rb1, CARF and SGK3.

Highlights

Cigarette smoke consists of a complex mixture of thousands of toxic chemicals and over 1015 reactive oxygen species [1]
We found miR-335-5p levels to be lower in the parenchymal lung fibroblasts of current smokers compared to those of ex-smokers, and this was validated in lung tissue
In bone-marrow derived human mesenchymal stem cells, the overexpression of miR-335-5p had an inhibitory effect on cell proliferation, migration, and differentiation [34]. This suggests that lower miR-335-5p levels as observed in current smokers and upon cigarette smoke extract (CSE) exposure in this study could result in enhanced proliferation, as well as in other cell types, such as fibroblasts

Summary

Introduction

Cigarette smoke consists of a complex mixture of thousands of toxic chemicals and over 1015 reactive oxygen species [1]. Oxidative stress caused by cigarette smoking can dysregulate cell function, and induce the damage and death of the cellular constituents of the lungs [2]. Several studies have shown smoking-induced changes in gene expression patterns in the lung. Aberrant gene expression signatures were reported in epithelial cells when comparing current smokers with never-smokers [3,4]. Marked changes in miRNA expression signatures have been reported in the bronchial airway epithelial cells, small airway epithelium, whole blood, and induced sputum supernatant of current smokers compared to never-smokers [6,7,8,9]. The altered expression signatures may be due to a direct effect of smoking, but can be caused by indirect effects such as smoking-induced aberrant DNA methylation patterns [10]. A previous study showed that when smokers quit smoking for three months, 65% of the miRNAs that were differentially expressed between current smokers and never-smokers return to the expression level of never-smokers [7]

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