Analysis of microRNA and mRNA expression profiles highlights alterations in modulation of the MAPK pathway under octanal exposure

Highlights

• Microarray analysis identified 15 miRNAs that were differentially expressed in OC-exposed A549 human alveolar cells.

• Integrated analyses of miRNA and mRNA expression profiles identified significant miRNA–mRNA anti-correlations.

• GO analysis of 101 putative target genes showed that the biological category ‘MAPK signaling pathway’ was prominently annotated.

• Phosphorylation of p38 MAPK was increased in OC-exposed A549 cells.

Abstract

Previous environmental microRNA (miRNA) studies have investigated a limited number of candidate miRNAs and have not evaluated functional effects on gene expression. In this study, we aimed to identify octanal (OC)-sensitive miRNAs and to characterize the relationships between miRNAs and expression of candidate genes involved in OC-induced toxicity.

Microarray analysis identified 15 miRNAs that were differentially expressed in OC-exposed A549 human alveolar cells. Integrated analyses of miRNA and mRNA expression profiles identified significant miRNA–mRNA anti-correlations. GO analysis of 101 putative target genes showed that the biological category ‘MAPK signaling pathway’ was prominently annotated. Moreover, we detected increased phosphorylation of p38 MAPK in the OC-exposed group.

By integrating the transcriptome and microRNAome, we provide evidence that OC can affect MAPK-induced toxicity signaling. Therefore, this study demonstrates the added value of an integrated miRNA–mRNA approach for identifying molecular events induced by environmental pollutants in an in vitro human model

Introduction

Air pollution is known to be associated with health effects on the respiratory tract, such as asthma symptoms and bronchial hyperreactivity; although, the relationships between specific health effects and indoor air pollutants need to be specified (Bernstein et al., 2008). To clarify the relationship between indoor pollution and lung diseases such as inflammation, systemic toxicological data are needed in addition to in vitro experimental evidence.

Aldehydes are a group of volatile organic compounds (VOCs) that have the potential to be airborne risk chemicals. To increase the energy efficiency of buildings, air-exchange rates of indoor and outdoor air are decreased, which causes the accumulation of VOCs indoors. Aldehydes are emitted from many materials, such as building materials, furnishings, paints, varnishes, waxes, solvents, detergents, cleaning products, cooking oil, and fried foods (Baumann et al., 2000, Nazaroff and Weschler, 2004, O’Brien et al., 2005). Despite their usefulness, aldehydes have been reported to be harmful with mutagenic and carcinogenic effects. Recent studies showed that aldehyde exposure leads to inflammation, oxidative stress, and immunomodulation in the airways and is associated with airway inflammatory disorders such as asthma and chronic obstructive pulmonary disease (COPD) (Fujimaki et al., 2007, Jung et al., 2007). Dissecting the in vitro effects of aldehyde exposure on pulmonary cells is therefore of importance to our understanding of initiating mechanisms in pulmonary disease.

Mitogen-activated protein kinases (MAPKs) are central regulators of inflammatory responses to exogenous (e.g., infections) and endogenous (tissue injury) insults (Coulthard et al., 2009, Kim et al., 2008). MAPKs contribute to the inflammatory responses induced by environmental toxicant exposure, endotoxins and oxidative stress through the activation and release of proinflammatory cytokines/chemokines, posttranslational regulation of the genes encoding them, and activation of inflammatory cell migration (Perdiguero et al., 2011, Kim et al., 2009). Modifications of inflammatory gene expression through the MAPK signaling pathway has been demonstrated in formaldehyde (FA)-exposed BEAS-2B human lung cells, possibly reflecting effects on pulmonary inflammation (Lecureur et al., 2012). However, the molecular pathways that determine the systemic changes in gene expression after exposure to other aldehydes such as octanal (OC) are still largely unknown.

Previous research has shown altered inflammatory-responsive gene expression patterns in lung cells exposed to several aldehydes (Lee et al., 2012). These changes in gene transcript profiles, which likely translate to changes in protein levels, could arise from altered microRNA (miRNA) expression. miRNAs are an abundant class of regulatory molecules that have received scientific attention for their ability to alter mRNA abundance.

miRNAs represent an important group of noncoding RNAs and play an important role in translational regulation. They are 18–26 nucleotides in length and are generally transcribed in the nucleus by RNA polymerase II (Bartel, 2009). Increasing evidence shows that several alterations in miRNA profiles are involved in developmental timing and patterning, differentiation, and organogenesis, as well as disease pathology. This type of profiling has been proposed as a novel method of classifying diseases (Lu et al., 2005, Volinia et al., 2006). Also, miRNAs may play a significant role in responses to xenobiotic chemicals and their role in causing various health-associated problems and ailments. Fukushima et al. showed that exposure of rats to liver toxicants such as acetaminophen and carbon tetrachloride caused alteration in the expression of various miRNAs (Fukushima et al., 2007). In another report, tamoxifen, a potent hepatocarcinogen, was shown to increase the expression of several miRNAs associated with oncogenes (Pogribny et al., 2007). Other reports have suggested that drug-metabolizing enzymes such as CYP family genes are targeted by certain miRNAs (Komagata et al., 2009, Tsuchiya et al., 2006). These reports suggest that miRNAs may regulate the toxicity mediated by environmental chemicals.

Mammalian miRNAs regulate gene expression at the post-transcriptional level. miRNAs predominantly decrease target mRNA levels through destabilization (Guo et al., 2010). It has been estimated that ∼30% of protein-coding genes contain potential miRNA-binding sites in their 3’- untranslated regions (3’-UTRs) and may therefore be under miRNA control (Lynam-Lennon et al., 2009, Sun and Tsao, 2008, Wang et al., 2008). Therefore, coherent miRNA–mRNA expression changes, defined as a negative correlation between miRNA expression and expression of mRNAs that are predicted targets of the miRNA(s), can occur (Liu and Kohane, 2009). miRNA–mRNA interactions may explain some effects on cellular physiology and even dramatic effects on cell phenotype (Lafferty-Whyte et al., 2009). To gain deeper insight into the mechanism of toxicity, it is important to identify and characterize the profiles of miRNAs involved in responses to specific classes of toxicants in conjunction with their impact on gene expression levels.

In the present study, we hypothesized that miRNA–mRNA interactions are involved in the MAPK signaling pathways activated by environmental pollutants. The objective of our study was to investigate the modulation of miRNA–mRNA networks following exposure to OC and to investigate MAPK activity using an in vitro system. miRNA expression profiles and global gene expression changes were investigated in A549 human alveolar epithelial cells. By performing bioinformatic analyses, we found that OC induces miRNA–mRNA interactions that are closely related to MAPK signaling pathways.