MicroRNA expression profiling defines the impact of electronic cigarettes on human airway epithelial cells

While all forms of tobacco exposure have negative health effects, the significance of exposure to electronic cigarettes (eCig) is not fully understood. Here, we studied the global effects of eCig on the micro RNA (miRNA) transcriptome in human lung epithelial cells. Primary human bronchial epithelial (NHBE) cells differentiated at air-liquid interface were exposed to eCig liquid. Exposure of NHBE to any eCig liquid resulted in the induction of oxidative stress-response genes including GCLM, GCLC, GPX2, NQO1 and HO-1. Vaporization of, and/or the presence of nicotine in, eCig liquid was associated with a greater response. We identified 578 miRNAs dysregulated by eCig exposure in NHBE, and 125 miRNA affected by vaporization of eCig liquid. Nicotine containing eCig vapor displayed the most profound effects upon miRNA expression. We selected 8 miRNAs (29A, 140, 126, 374A, 26A-2, 147B, 941 and 589) for further study. We validated increased expression of multiple miRNAs, including miR126, following eCig exposure. We also found significant reduction in the expression of two miR126 target genes, MYC and MRGPRX3, following exposure. These data demonstrated that eCig exposure has profound effects upon gene expression in human lung epithelial cells, some of which are epigenetically programmed at the level of miRNA regulation.

in primary human airway epithelial cells 21 . In addition, exposure of human airway epithelial cells to eCig aerosols induces the secretion of inflammatory cytokines, IL-6 and IL-8 22 .
Exposure of the lung epithelial cells to cigarette smoke induces a variety of effects directly measurable at the cellular and molecular level [23][24][25] . Increased oxidative stress is a major driving mechanism in the pathophysiology of smoking-related lung diseases such as COPD 18,26 . The presence of a large amount of free radicals in cigarette smoke 27 disturbs the redox balance in the lungs, leading to increased oxidative burden 28 . In COPD, antioxidant capacity is reduced and oxidative stress persists even after the cessation of smoking due to the continued production of reactive oxygen species from endogenous sources, contributing to COPD comorbidities such as cardiovascular diseases, metabolic syndrome and lung cancers 26 . Conversely, very little is known about the role of eCigs in oxidative lung damage. A recent report suggested that the oxidative stress inducing capacity of eCigs depends on their flavor additives, with flavors containing sweet or fruit flavors being stronger oxidizers than tobacco flavors 22 . eCig liquids have been shown to profoundly alter the metabolome of bronchial epithelial cells partly similar to cigarette smoke condensate, and the use of antioxidants can partially counteracted this response 10 . Further, soluble components of eCigs, including nicotine, cause loss of lung endothelial barrier function, due to oxidative stress and inflammation 29 . Furthermore, exposure of bronchial epithelial cells to nicotine induces apoptosis and senescence via ROS mediated autophagy-impairment and could act as a potential mechanism for COPD-emphysema pathogenesis 30 .
Genome-wide transcriptional analysis of microRNA expression in eCig treated NHBE. We were particularly interested in exploring epigenetic regulatory mechanisms induced by eCig exposure. Therefore, we transcriptionally profiled 2541 miRNA from NHBE which were either treated with non-vaporized liquid without (LIQ-NONIC), with nicotine (LIQ-NIC), vaporized/condensed liquid without (VAP-NONIC) or with nicotine (VAP-NIC), cigarette smoke condensate (CSC), or untreated control cells (CTL). Hierarchical cluster analysis of the data set indicated a clear segregation among cells treated with any eCig liquid as compared to other groups (Fig. 3A). Similar clusters were observed using non-parametric bootstrap analysis.  Table 1. The expression profiles for these miRNAs is presented tin Fig. 3B. SAM-Seq (at median FDR = 0) identified 125 microRNAs as significantly differentially expressed between cells treated with any vaporized liquid (n = 6) compared to any non-vaporized liquid (n = 6) as listed in Supplemental Table 2. The expression profiles for these miRNAs is presented in Fig. 3C. miRNA-mRNA regulation by electronic cigarette exposure. Based upon the magnitude of response, and biological interest, we attempted to validate differential expression for eight miRNAs predicted to be significantly affected by treatment with any eCig liquid (Fig. 4A). Similar to increased expression predicted by RNA-Seq analysis, we found a significant increase in the expression of MIR26A-2-3P (5.9 fold, p < 0.05), MIR126-5P (12.6 fold, p < 0.05), MIR140-5P (5.6 fold, p < 0.05), MIR29A-5P (6 fold, p < 0.05), MIR374A-3P (10.2 fold, p < 0.05) and MIR147B (3.6 fold, p < 0.05) by qPCR (Fig. 4B). Conversely, expression of MIR941 (50%, p < 0.05) and MIR589-5P (69%, p < 0.05), which were predicted by RNA-Seq analysis to be down regulated, were not validated by qPCR.
Based upon these data, we chose to further explore the induction of MIR126-5P, and test whether this results in inhibition of its target genes in response to eCig treatment. MIR126-5P is significantly induced by eCig liquid, particularly following vaporization (Fig. 5A). VAP exposure increased MIR126-5P expression (1.6 fold, p < 0.05) as compared to NON VAP exposure (15.6 fold in VAP vs 9.6 fold in NON VAP, p < 0.05). A modest increase in expression of MIR126-5P noted upon CSC treatment (2.6 fold) did not reach statistical significance.

Discussion
MiRNAs are small RNA molecules (21-25 nucleotides long) that regulate gene expression post-transcriptionally, either by mRNA degradation or inhibition of translation or both 33 . MiRNAs are predicted to regulate approximately 60% of protein-coding genes in human 34 . MiRNAs have been recognized as modulators of smoking-induced gene expression changes in human airway epithelium 35 , and dysregulation of miRNA expression are associated with many pulmonary diseases including COPD 35,36 . Electronic cigarettes have become very popular in recent years, with an estimated 13 million people using globally 2,13 . ECigs may help reduce tobacco smoking 3,4,37 , however the safety of eCigs has yet to be fully appreciated 10,13,38,39 . In the present study we demonstrate that exposure to eCig liquid regulates oxidative stress response genes and numerous miRNAs in human lung cells.
Transcriptional profiling for the assessment of global changes in expression of coding and non-coding RNA species associated with cigarette smoke exposure, in vitro or in vivo, have been reported 35,[40][41][42][43][44][45] . Similar analyses of the effects of eCig exposure could help to assess the potential health effects 46 . Transcriptome analysis has identified gene expression profiles in human bronchial epithelial cells upon eCig vapor and mainstream-smoke from tobacco cigarette exposure 47 . Further, transcriptional profiling for immune and inflammatory-response genes in nasal epithelial cells from eCig users has been reported 48 recently. However, no comprehensive analysis of eCig effects upon lung epithelial cell miRNA expression been previously reported. Our results indicate that eCig exposure induces the expression of oxidative stress response genes, and causes dysregulation of numerous miRNAs in human bronchial epithelial cells in vitro.
In vitro and in vivo studies have shown that miRNAs play a crucial role in cellular response to stress as well as cell growth and death 35,36,49,50 . The airway epithelial miRNA transcriptome changes in response to cigarette smoke exposure 35,49 , and miRNAs have been implicated in COPD pathology 35,49,51 . A main goal of this study was to determine the global effects of eCig exposure upon miRNA expression, and test whether miRNAs alter expression of their target mRNA in eCig-exposed lung epithelial cells. We have identified 578 miRNAs as differentially expressed with eCig treatment. Interestingly, vaporization of eCig liquid resulted in dysregulation of 125 miRNA compared to non-vaporized liquid. While this is the first study to report the miRNA transcriptome profiling in response to eCigs, two recent studies describe global changes in mRNA expression under similar conditions 47,48 .
Among the eight selected miRNAs, we focused our interest upon MIR126, which is located in chromosome 9q34.3 within the host gene encoding for epidermal growth factor like-7 (EGFL-7) 52, 53 . It is highly expressed within highly vascularized adult tissues like the lung, heart, and kidney 54,55 . Its expression is decreased in lung tumor and appears to be regulated epigenetically by demethylating agents 56,57 . MIR126 has been shown to regulate various biological processes including inhibition of invasion in non-small cell lung carcinoma 52 , to affect mitochondrial energy metabolism resulting in malignant mesothelioma tumor suppression 58 , and to regulate leucocyte adhesion and transmigration across the endothelium 59 . Soluble components of eCig vapor, including nicotine, have been reported to cause loss of lung endothelial barrier function, associated with oxidative stress and inflammation 29 . We found increased expression of MIR126-5P in all conditions tested, with the greatest effect being in cells exposed to vaporized eCig liquid. Importantly, increased expression of MIR126-5P was associated with reduced expression of its target genes, MYC and MRGPRX3.
Oxidative stress has been recognized as a major driving mechanism in the pathophysiology of COPD 18,26,60 . In COPD, there is an imbalance between antioxidant capacity and oxidative stress due to excessive generation of reactive oxygen species (ROS) 26 . In the present study we show that eCigs are potent inducers of OxS response genes in human bronchial epithelial cells, particularly those that contain nicotine. Similar to our findings, recent reports also suggest that eCigs induce oxidative stress, depending on the presence of flavor additives or nicotine 22,61 . ECig aerosols have also been shown to contain oxidants and copper nanoparticles, inducing mitochondrial stress evident by elevated levels of mitochondrial ROS from human lung fibroblasts 22,62 .
We specifically found increased expression of genes (GCLC and GCLM) that catalyze the production of the cellular endogenous antioxidant glutathione (GSH). Of all antioxidants, higher concentrations of GSH have been shown to be present in the lungs and these levels are even further increased in smokers 63 . Likewise, we found eCigs increase the expression of glutathione peroxidases (GPX), the primary antioxidant enzymes that scavenge and detoxify hydrogen peroxide and organic hydroperoxides, which are also increased in the lungs of cigarette smoke-exposed mice 64 . We have also found increased expression of NQO1 and HO1. NQO1 is a flavoprotein, which exerts either antioxidant or pro-oxidant properties depending on the quinone substrate. HO1, the inducible isoform of heme oxygenase, is a cytoprotective enzyme that plays a critical role in the defense against oxidative and inflammatory insults in the lung 65 . The basic leucine zipper transcription factor, Nrf2 plays an important role in the transcriptional regulation of oxidative stress response genes. However, eCigs did not induce the expression of Nrf2 in bronchial epithelial cells. Nuclear translocation of Nrf2, not necessarily expression levels, have been shown to be important in the transcriptional induction of antioxidant and phase II detoxification genes in different cells 25 .
The relationship between eCig-induced oxidative stress and miR dysregulation is currently unclear. In the current study, we used oxidative stress gene expression responses as a measure (positive control) for previously reported effects of eCig liquid, and further explored the effects of vaporization and nicotine content on these responses. We were also interested in potential "epigenetic" effects of eCig liquid exposure in lung epithelial cells. Therefore, we surveyed miRNA dysregulaiton by eCig liquid at the genome-wide level. We queried multiple databases (mirPath 66 , Ingenuity Pathway Analysis) for functional analysis of the differentially expressed miRNAs we identified, in order to determine if they (as a set) had pre-existing relationships with regulating oxidative stress response genes. We did not find any associations; most likely due to lack of sufficient annotations for function of miRs in existing databases.
In summary, we have profiled global miRNA expression in the bronchial epithelium exposed to electronic cigarettes. Our findings suggest that exposure to eCigs induces oxidative stress response gene expression and causes dysregulation of many miRNAs in bronchial epithelial cells. We have further demonstrated anti correlation between MIR126-5P and its target genes, MYC and MRGPRX3. MicroRNA profiles observed from this study might therefore serve as biomarkers for defining eCig exposure, as well as their potential pathophysiological effects. Cell Culture. NHBE cells were cultured as previously described, with modifications 19 , and used between passages 2 and 4. Briefly, NHBE cells were expanded in bronchial epithelial basal medium (BEBM, Lonza) containing bovine pituitary extract, hydrocortisone, human recombinant epidermal growth factor (25 ng/mL), epinephrine, insulin, triiodothyronine, transferrin, gentamicin, Amphotericin B, retinoic acid, and BSA. Cells were transferred to rat tail collagen-coated Transwell inserts (12 well PET membrane, 0.4 µm pore size, 12 mm diameter) for Air-Liquid Interface culture (ALI). When the cells in the transwell plate reached confluence (10-12 days), with appropriate resistance (200-300 Ohms/cm 2 ), cells were transferred to ALI by removing the apical medium, and cultured for additional 14 days. For growth of cells on transwell plates, the basal medium was modified to a 1:1 mixture of BEBM/Dulbecco modified Eagle medium (DMEM) with high glucose containing the same supplements, except with a lower concentration of human recombinant epidermal growth factor (0.5 ng/mL). Upon transition to ALI, cells were maintained using PneumaCult-ALI medium (Stemcell technologies, Canada) containing PneumaCult-ALI maintenance supplements and hydrocortisone, according to manufacturer's instructions. The medium was replaced every 48 hrs. Figure 1 presents the apparatus used to prepare electronic cigarette condensate. Briefly, eCig liquid, without or with nicotine (0% or 2.4% w/v nicotine, respectively), was added to a Nautilus Mini tank system with 1.8 ohm BVC atomizer, and connected to a charged electronic smoking device. Using a custom tube assembly, 40 ml "puffs" were drawn with a 60 ml syringe, with each puff lasting for 4 seconds. The puffs were drawn at 7.5 W output power settings of the electronic smoking device. The eCig vapor condensed in a 50 ml falcon tube chilled above a liquid nitrogen/dry ice bath. Using this assembly, 1000 ml eCig vapor yielded approximately 20 µl of condensate. Freshly prepared eCig condensates were used for every experiment. Cells were treated with eCig liquid, vaporized and condensed eCig liquid (2%) or cigarette smoke condensate 19 (Murthy pharmaceuticals, City, State), baso-laterally for 48 hrs. Cells treated with medium alone, served as control. This concentration was selected based upon preparatory studies indicating graded dose-response effects of concentrations between 0.1-2.0% upon oxidative stress response gene expression.

RNA Isolation and Reverse Transcription and Real-Time Quantitative RT-PCR (qPCR). DNA
free-total RNA was isolated from cultured cells, using the Absolute RNA miRNA kit. For mRNA analysiss, total RNAs were reverse-transcribed using the iScript cDNA Synthesis Kit. qPCR analysis was performed using using SYBR Green chemistry. Quantitative real-time PCR Gene expression levels were calculated relative to Ppia (cyclophilin A) using the ddCT method as we have previously described 19,67 . Our choice of Ppia was based upon results of our previous analysis of gene expression in lung tissues and cells, from different age groups and conditions, where we found Cyclophillin A (PPIA) to be highly stable across all conditions. Consistent with our observations, PPIA was found to be a better candidate housekeeping gene than GAPDH and ACTB, when considering variability of expression 68,69 . In fact, in prior human studies 42 we have found that PPIA alone provided equivalent sensitivity and resolution compared to multi-gene normalization approach GeNorm 70 . Primer sequences were selected from PrimerBank (http://pga.mgh.harvard.edu/primerbank/). For miRNA analysis, total RNAs were reverse transcribed using Taqman miRNA reverse transcription kit with minor modifications. For a 15 µl reaction, total RNA (100ng) was combined with RT master mix (0.2 µl dNTP, 0.19 µl RNase-Inhibitor, 1 µl microRNA specific primer, 1 µl multiscribe RT enzyme and 1.5 µl buffer). qPCR analysis was performed using microRNA specific primers supplied with Taqman MicroRNA assay. U6 snRNA was used as normalization control. Small non-coding RNA U6 is a widely used normalization control for microRNAs, has been reported to have stable expression, and was used at the recommendation of the manufacturer of the TaqMan-based miRNA quantitation assay reagents (Thermo Fisher Scientific, Foster City, CA). Gene expression levels were calculated using the ddCT method as we have previously described 19,67 . Statistical analysis for differences in gene expression by qPCR was performed using either parametric (student's T) or non-parametric (Mann-Whitney U) tests of the mean. microRNA sequencing. Sequencing was performed on cDNA libraries generated using 1000 ng of total RNA from each sample. cDNA quantity was determined with the Qubit Flourometer (Life Technologies, Grand Island, NY) and quality was assessed using the Agilent Bioanalyzer 2100 (Santa Clara, CA). Library construction was performed using the TruSeq Small RNA Sample Preparation kit (Illumina, San Diego, CA). Libraries were quantified with the Qubit Flourometer (Life Technologies, Grand Island, NY) and quality was assessed using the Agilent Tape Station (Santa Clara, CA). Libraries were sequenced (single end reads) on the Illumina HiSeq2500 (Illumina, San Diego, CA) to generate 50 million reads/sample. MiRNA reads were aligned and mapped using miRGE 71 , and summarized at the raw reads level using the following components: Cutadapt for sequence read filtering and adapter trimming, and Bowtie for sequence read alignment to known mature human microRNA sequences. Raw reads obtained from each alignment algorithm were normalized using reads per million (RPM) bases. Hierarchical clustering, using Euclidean distance and average linkage, with non-parametric bootstrap was used to look for underlying unanticipated association among samples 72 . Differential gene expression was assessed by SAM-Seq 73, 74 which implements an FDR based approach for correction for multiple testing 75 .