ORIGINAL RESEARCH Expression of microRNA-93 and Interleukin-8 during Pseudomonas aeruginosa–Mediated Induction of Proinflammatory Responses Enrica Fabbri1, Monica Borgatti1, Giulia Montagner1, Nicoletta Bianchi1, Alessia Finotti2, Ilaria Lampronti1, Valentino Bezzerri3, Maria Cristina Dechecchi3, Giulio Cabrini3, and Roberto Gambari1,2 1

Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, and 2Biotechnology Center, University of Ferrara, Ferrara, Italy; and 3Laboratory of Molecular Pathology, Laboratory of Clinical Chemistry and Haematology, University-Hospital, Verona, Italy

Abstract In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3–1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 39-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated

MicroRNAs (miRs) are an evolutionarily conserved family of endogenous noncoding RNAs, approximately 19 to 25 nucleotides long, that play a posttranscriptional gene expression regulatory role in animals and plants by sequence-selective targeting of mRNAs (1, 2), inducing translational repression or mRNA degradation (3, 4). Considering that a single miR can target several mRNAs and that a single mRNA might contain signals for molecular recognition by several miRs, more than 60% of mammalian mRNAs are targets of microRNAs (2), leading to the control of

using three luciferase vectors, including one carrying the complete 39UTR region of the IL-8 mRNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3–1, CuFi-1, and NuLi-1 cells by pre–miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-kB–dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93. Keywords: microRNA; inflammation; lung; cystic fibrosis

highly regulated processes, such as differentiation, cell cycle, and apoptosis (1–4). Few reports are available on microRNA expression and proinflammatory pathways in respiratory tissues, although microRNA expression has been examined in lung tumors (5) and under a variety of inflammatory stimuli (6–8). For instance, Fang and colleagues reported that miR-10a regulates the proinflammatory phenotype of endothelium in vitro and in vivo and found that the inflammatory biomarkers monocyte chemotactic protein 1, IL-6, IL-8,

vascular cell adhesion molecule 1, and E-selectin were up-regulated after knockdown of miR-10a (9). O’Connell and colleagues published a summary of advancements in miRNAs and their connection to multiple clinic manifestation of inflammation (10). Other microRNAs involved in the regulation of inflammation have been proposed, such as miR-146a/b, miR-132, miR-155, and miR-126 (11–14). Finally, the effects of microRNAs miR-138, miR-145, and miR-494 on the regulation of the expression of wild-type (WT) and of DF508 cystic fibrosis transmembrane conductance

( Received in original form April 5, 2013; accepted in final form January 12, 2014 ) This work was supported by Fondazione Cariparo (Cassa di Risparmio di Padova e Rovigo), Consorzio Interuniversitario di Biotecnologie (CIB) (R.G.); by Telethon (contract GGP10214); by COFIN-2009 (R.G.); by Italian Cystic Fibrosis Research Foundation grants FFC 17#2010, FFC#19/2011, and FFC 1#2012 (R.G.); by Associazione Italiana Ricerca sul Cancro (AIRC, contract IG13575) (R.G.); by Associazione Veneta per la Lotta alla Talassemia (AVLT); by a CIB fellowship (E.F.); and by the Italian Cystic Fibrosis Research Foundation (V.B.). Correspondence and requests for reprints should be addressed to Roberto Gambari, Ph.D., Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, Via Fossato di Mortara, 74, 44121 Ferrara, Italy. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 50, Iss 6, pp 1144–1155, Jun 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0160OC on January 16, 2014 Internet address: www.atsjournals.org

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ORIGINAL RESEARCH regulator (CFTR) were recently reported (15, 16). Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease resulting from mutations of the CFTR gene, which encodes for a chloride channel transcript expressed in specialized epithelia of the airways and in different exocrine tissues (17). CFTR conducts Cl2, HCO32, and other anions (18) regulating the composition and volume of airway surface liquid. Although defective CFTR affects many organs (e.g., lungs, pancreas, liver, and the reproductive system), 85% of the mortality results from progressive airway tract obstruction ultimately resulting in respiratory failure (19–21). The most important clinical complication in the pulmonary tissue of patients with CF is persistent infection and exaggerated inflammatory response (22). Inflammation starts in the early phases of the disease (even in the absence of infection), is further amplified by recurrent bacterial infections of the epithelial surface (e.g., by Hemophilus influenzae and Staphylococcus aureus), and is followed by chronic bacterial colonization with highly antibiotic-resistant Pseudomonas aeruginosa communities (23). To determine the role of miRNA expression in P. aeruginosa dependent upregulation of the IL-8 proinflammatory gene in bronchial epithelial cells, CF bronchial epithelial IB3–1 and CuFi-1 cells and the non-CF bronchial NuLi-1 cells were infected with P. aeruginosa.

Materials and Methods Cell Lines and PAO1 Strain

IB3–1, obtained from LGC Promochem, Europe, is a human bronchial epithelial cell line immortalized with adeno12/SV40, derived from a patient with CF with an F508 del/W1282X mutant genotype (24). CuFi-1 and NuLi-1 cells (a generous gift of A. Klingelhutz, P. Karp, and J. Zabner, University of Iowa, Iowa City, IA) derive from human bronchial epithelium of a patient with CF (CuFi-1, F508 del/F508 del CFTR mutant genotype) or from a subject without CF (NuLi-1, WT CFTR) and have been transformed by the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 E6 and E7 genes (25). Cell culture conditions are described in the online

supplement. PAO1, a prototypic laboratory strain of P. aeruginosa, was provided by A. Prince (Columbia University). Cells were treated with PAO1 (or heat-inactivated PAO1) as described elsewhere (26, 27). MicroRNA Analysis

For microRNA expression study two approaches were followed: microRNA profiling and quantitative RT-PCR (RTqPCR). RNA analysis with microRNA microarray chips (Agilent Technologies, Santa Clara, CA) has been carried on using a platform containing 470 human miRNA probes for mature and precursor microRNAs (28). Raw data were normalized and analyzed by GeneSpring GX software version 7.3 (Agilent Technologies). Reactions for microRNA quantification were performed using the TaqMan MicroRNA Reverse Transcription Kit, the TaqMan MicroRNA Assay Kit (Applied Biosystems, Foster City, CA), and the 7700 Sequence Detection System version 1.7 (Applied Biosystems). Relative expression was calculated using the comparative cycle threshold method and the human U6 and let-7c as reference genes. IL-8 and Minichromosome Maintenance Complex Component 7 mRNA Content

Total RNA (300 ng) was reverse transcribed and then amplified with iQ SYBR Green Supermix or TaqMan Assay kit using the CFX instrument (Bio-Rad, Hercules, CA). Minichromosome maintenance complex component 7 (MCM7) gene expression was detected with the assay “Hs 0028518_m1,” and its reference gene was detected with the 18S assay (Applied Biosystems). Primer sequences for IL-8 mRNA detection were forward 59-GTG CAG TTT TGC CAA GGA GT-39 and reverse 59-TTA TGA ATT CTC AGC CCT CTT CAA AAA CTT CTC-39 (Sigma-Genosys, The Woodlands, TX). The expression levels of mRNAs were calculated after normalization with the 18S and RPL13a gene using the comparative cycle threshold method. Bio-plex Analysis

Cytokines, chemokines, and growth factors released in the tissue culture supernatants were measured using the Bio-plex Pro Human Cytokine Grp I Panel 7-plex or 27-plex kits (Bio-Rad) (29, 30). Technical details are reported in the online

Fabbri, Borgatti, Montagner, et al.: microRNA-93 and IL-8 mRNA in CF

supplement. The levels of multiple transcription factors within nuclear extracts, obtained from stimulated cells, were analyzed by BioSource’s Transcription Factor (TF) Assays (Biosource International, Inc., Camarillo, CA). Nuclear extracts were prepared using the Nuclear Extraction Kit (Biosource International) and quantified using the Bradford assay. The assay was performed as reported by the manufacturer and as briefly described in the online supplement. The samples were analyzed using a Bio-plex instrument. Reporter Gene Assay

For these experiments, we used the IL-8 (NM_000584)-39-UTR-pMirTarget Vector luciferase plasmid (OriGene Technologies, Rockville, MD). In addition, we produced an empty vector without the IL-8 39-UTR sequence and a mutated IL-8 39-UTR-pMir vector mutagenized at the level of the miR-93 binding site. The generation of these two vectors is described in the online supplement. IB3–1 cells were seeded in 12-well plates (1.0 3 105 cells per well). The next day, cells were transfected using 1 ml X-tremeGENE HP DNA (Roche, Basel, Switzerland) for 1 mg of plasmid vector, pre–miR-93, antagomiR-93, and relative negative controls (Ambion, Austin, TX) at a final concentration of 1 mM. The lysates were prepared 48 hours after transfection, and luciferase activity was measured by using the neolite Reporter Gene Assay System (PerkinElmer, Waltham, MA) with a VICTOR3 Multilabel Counter model 1420–051 (PerkinElmer) using the red fluorescent protein as internal control. Predicted MicroRNA Target

Different online algorithms (PicTar, TargetScan 6.2, miRBase, miRNA.org) were used to identify putative binding sites between the seed region of miRNA and the mRNA of potential target genes. Pre-miR and AntagomiR Transfections

IB3–1, CuFi-1, and NuLi-1 cells were transfected with antagomiR-93, pre-miR93, and the miR negative controls (Ambion) complexed with siPORT NeoFX (Life Technologies, Carlsbad, CA). Infection with PAO1 was performed 24 hours after transfection. After a further 24 hours, cell supernatants were collected, and total RNA was extracted and immediately converted to cDNA. 1145

ORIGINAL RESEARCH Statistics

Results are expressed as mean 6 SEM. Comparisons between groups were made using the paired Student’s t test and a oneway ANOVA. Statistical significance was defined as significant at P , 0.05 and highly significant at P , 0.01.

Results Characterization of Expression of Proinflammatory Genes in CF IB3–1 Cells Infected with P. aeruginosa

The effect of P. aeruginosa infection on the expression of proinflammatory genes was analyzed in the CF bronchial IB3–1 cell line (31–33). This is a well-established procedure reproducibly showing that shortterm exposure of cells to the P. aeruginosa lab strain PAO1 increases IL-8 mRNA by several fold compared with basal levels of uninfected cells; this closely mimics the huge amount of the neutrophil chemokine IL-8 usually detected in CF respiratory tissue and secretions, a hallmark of CF lung pathology. In addition to IL-8 mRNA, PAO1 induces a relevant expression of other proinflammatory genes, such as GRO-g, GRO-a, IL-6, IL-1b, and ICAM-1, whereas low increases of IP-10, RANTES, MIP-1a, TNF-a, IFN-g, TGF-b, and IL-10 mRNAs are usually observed under these experimental conditions (31). Here we reproduced the increase of IL-8 protein release from IB3–1 cells exposed to PAO1 for short time (Figure 1A). These

data provide clear evidence that IL-8 mRNA transcription and IL-8 protein secretion occur soon after infection with PAO1, as reported in several papers from other and our research groups. Figure 1B shows the location of microRNAs potential target sites identified, within the 39-UTR of the human IL-8 mRNA, by in silico analyses. The prediction analyses indicated at least 34 different microRNAs that could possibly target this IL-8 mRNA region, supporting the concept that the expression of IL-8 gene could be posttranscriptionally regulated by several microRNAs, in addition to the well-characterized transcriptional control (33). MicroRNA Profiling Analysis

The microarray-based screening was performed using the Agilent microRNA chip and LNA-modified oligonucleotides, which contains 470 miR probes and RNA isolated from uninfected IB3–1 cells or PAO1infected cells. Out of 470 human miRNAs, 320 were found not expressed or expressed at extremely low levels in the two samples analyzed here. Figure 2A reports the overall analysis performed on the 150 miRNAs found expressed in at least one sample, using a color-bar approach (Figure 2, left). When the analysis was restricted to microRNAs showing at least a 2-fold change in the level of expression, nine miRNAs were found to be down-modulated (Table 1). The microarray analysis was used to verify whether some of the 34 microRNAs

predicted in silico to interact with the 39UTR sequence of IL-8 mRNA (Figure 1B) were down-regulated upon exposure of the cells to PAO1, which is known to dramatically increase IL-8 gene transcription. The results depicted in Figure 2B indicate miR-93 and miR-494 as the most interesting microRNAs in terms of changes of expression. The decrease of the content of these microRNAs was confirmed in several validation experiments using RTqPCR as the analytical method (compare the data of Figure 2C with those of Figure 2A). However, in all the experiments performed, inhibition of miR-494 was found lower than inhibition of miR-93 expression when IB3–1 cells were infected with PAO1. Figure 3A shows the potential interactions of miR-93 within the 39-UTR sequences of the IL-8 mRNA at the level of RNA-induced silencing complex. When a secondary structure of IL-8 39-UTR is generated, the lowest energy secondary structure of miR-93 is potentially able to interact with an IL-8 mRNA sequence contained in a singlestranded loop structure. On the contrary, a miR-494 sequence at the 59 end interacts only with a stem IL-8 39-UTR structure. For these reasons, we focused on miR-93 as a microRNA possibly playing a role in the upregulation of IL-8 gene expression in P. aeruginosa–infected IB3–1 cells. The down-regulation of miR-93 after P. aeruginosa infection was also confirmed in CF CuFi-1 and non-CF NuLi-1 bronchial epithelial cell lines (see Figure E1 in the online supplement).

Figure 1. (A) Bio-plex analysis of the release of IL-8 in the supernatant of IB3–1 cells alone and infected with heat-inactivated Pseudomonas aeruginosa. Supernatant from cell culture was collected after 4 hours, and IL-8 protein was determined by Bio-plex assay. In IB3–1 cells treated with PAO1 (white bar), IL-8 content increases several-fold compared with basal levels of uninfected cells (black bar). Data represent the average 6 SD (n = 3). (B) Location of microRNA (miR) target sequences (arrows) within the 39-UTR region of the IL-8 mRNA.

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Figure 2. Cluster analysis of miRNA expression profile using miR microarray chips. (A) When samples from uninfected IB3–1 cells and IB3–1 cells infected with PAO1 are compared with the 150 miRNAs found expressed in at least one sample, only miR-93 and miR-494 are identified as the most interesting down-regulated microRNAs. (B) Comparison between a list of the most relevant nine down-regulated miRNAs from microarray analysis and the 34 microRNAs possibly interacting with the 39-UTR sequence of IL-8 mRNA. miR-93 and miR-494 are in common between the two groups. (C) miR93, miR-494, and IL-8 mRNA expression determined by quantitative RT-PCR analysis in PAO1–infected cells and compared with that found in uninfected IB3–1 cells. Data represent the average 6 SD (n = 3).

The Sequences of IL-8 mRNA Recognized by miR-93 Are Conserved through Molecular Evolution

The conclusions of the first set of analyses shown in Figures 2 and 3 clearly indicate that (1) a putative miR-93 binding site is

located at nucleotide position 581 in the 39UTR sequence of the human IL-8 mRNA; (2) miR-93 is highly expressed in IB3–1 cells; (3) miR-93 is down-modulated after P. aeruginosa infection of IB3–1 cells; and (4), in agreement with several published

Fabbri, Borgatti, Montagner, et al.: microRNA-93 and IL-8 mRNA in CF

reports, the miR-93 sequence is highly conserved in different species. A further indication of the possible roles of miR-93 in IL-8 mRNA regulation is the finding reported in Figure 3C demonstrating that the 39-UTR sequence of the human IL-8 1147

ORIGINAL RESEARCH Table 1: Down-Regulated microRNAs after Infection of IB3–1 Cells with Pseudomonas aeruginosa* Systematic Name hsa-miR-93 hsa-miR-423-3p hsa-miR-494 hsa-miR-149 hsa-miR-324-3p hsa-miR-342-3p hsa-miR-768-3p hsa-miR-92a hsa-miR-484

Fold of Observed Down-Regulation

Uninfected IB3-1 Cells (Normalized)

PAO-1–Infected IB3-1 Cells (Normalized)

16.8 3.7 3.1 2.5 2.4 2.4 2.2 2.1 2.1

6.2 2.9 3.2 4.4 4.3 3.6 6.3 8.3 4.2

2.1 1 1.6 3.1 3.1 2.4 5.1 7.2 3.2

*RNA from control and P. aeruginosa–infected IB3-1 cells was isolated and microarray analysis performed as described in MATERIALS AND METHODS.

mRNA potentially interacting with miR-93 is highly conserved throughout molecular evolution. Full homology with the human mRNA sequence occurs not only in the IL-8 mRNA sequence of Pan troglodytes and Macaca mulatta but also in evolutionary divergent species. The gene regions surrounding the miR-93 target site of the IL-8 39-UTR are, on the contrary, divergent (Figure 3C). This is clearly in favor of a strong role of this sequence in regulating IL-8 gene expression, as suggested in a different experimental cell system (34). Moreover, as shown in Figure 3D, the extent of miR-93/IL-8 mRNA recognition is similar to that reported for other already validated target mRNAs of miR-93, such as F3, AID, Intg-b8, PTEN, and FUS1 mRNAs (35–39), strongly supporting the hypothesis that IL-8 mRNA is a target of miR-93. Decrease of miR-93 in IB3–1 Cells Infected with P. aeruginosa Is Accompanied by a Decrease of Transcription of the MCM7 Gene

miR-93 is an intragenic microRNA hosted by the gene coding the MCM7 protein (Figure 3A) (34). Accordingly, to further study the regulation of miR-93 gene expression in PAO1-infected IB3–1 cells, MCM7 mRNA was analyzed. To this aim, we repeated the PAO1 infection of IB3–1 cells and performed RT-qPCR analyses of miR-93, MCM7 mRNA, and IL-8 mRNA (Figure 4A), obtaining the expected increase of IL-8 mRNA content and, conversely, a parallel down-regulation of miR-93 and MCM7 mRNA sequences. To obtain preliminary pieces of information on the possible mechanisms leading to the PAO1-mediated down-regulation of 1148

the MCM7 gene, we determined the transcription factor level by Bio-plex analysis (Figures 4B and 4C). This finding suggests that, unlike the well-known NF-kB up-regulation (33), the content of E2F-1 and MYC transcription factors (two major transactivating factors of the MCM7 gene) (40, 41) is lower in PAO1-infected IB3–1 cells, suggesting a transcriptional downregulation of the MCM7/miR-93 cluster after P. aeruginosa infection of IB3–1 cells. Effects of Treatment with AntagomiR-93 and Pre–miR-93 on 39-UTR/IL-8 Luciferase Reporter Vectors

To further confirm the role of miR-93 on IL8 gene expression, we first used the IL-8 39-UTR-pMirTarget Vector, containing the Luc-mRNA sequence, upstream of the IL-8 39-UTR. This vector contains also a red fluorescent protein sequence under the control of the cytomegalovirus promoter to normalize for transfection efficiency. Higher luciferase activity has been observed in IB3–1 cells transfected with antagomiR93 compared with cells treated with a negative control (Figure 5A). In addition, when transfection of IB3–1 cells is performed in the presence of pre–miR-93, a decrease of luciferase activity was found compared with IB3–1 cells transfected with a negative control (Figure 5B). To determine the direct downregulation (decrease in luciferase activity) after the overexpression of miR-93, IB3–1 cells were transfected with three luciferase vectors with different expression of the IL-8 39-UTR region: (1) the IL-8 39-UTRpMirTarget Vector (vector), (2) the vector deleted of the IL-8 39-UTR region (empty vector), and (3) the vector containing an

IL-8 39-UTR region in which the miR-93 site was mutagenized (MUT-vector), as described in the MATERIALS AND METHODS section. These IB3–1 cells were also transfected with pre–miR-93 or with a negative control. The results of these experiments are shown in Figures 5C–5E and clearly indicate that pre–miR-93 treatment down-regulates luciferase activity of the IL-8 39-UTR–pMirTarget Vector (Figure 5C) but not of the empty vector (Figure 5D) and, more importantly, of the IL8 39-UTR mutagenized vector (Figure 5E). These findings strongly suggest that miR-93 exerts its specific regulatory effect directly on its putative binding site present within the 39-UTR region of the IL-8 mRNA, which becomes insensitive to pre–miR-93 effects even when it is only mutagenized at these sites. Effects of AntagomiR-93 and Pre–miR93 on IL-8 Expression in IB3–1, CuFi-1, and NuLi-1 Bronchial Cell Lines

To determine whether mimicking PAO1 down-regulation of miR-93, by using an antagomiR against miR-93, leads to induction of IL-8, IB3–1 cells were transfected with antagomiR-93, and the expression of miR-93 was analyzed by RTqPCR (Figure 6A). In addition, IL-8 mRNA content (Figure 6B) and IL-8 secretion (Figure 6C) were analyzed by RTqPCR and Bio-plex assays, respectively. The results demonstrate that antagomiR-93 reduces the miR-93 accumulation in IB3–1 cells (Figure 6A). In addition, Figure 6 demonstrates an increase of IL-8 mRNA content (Figure 6B) and a release of IL-8 (Figure 6C) in these antagomiR-93–treated IB3–1 cells, fully in agreement with the hypothesis of an involvement of miR-93 on IL-8 gene expression in CF cells. The decrease of miR-93 after treatment with antagomiR-93 is not unexpected, as found also by ours and other laboratories in different model systems (28, 42–44), and might be explained by a possible inhibitory action on miRNA processing. Figures 6D–6F show experiments in which pre–miR-93 has been transfected in IB3–1 cells before infection with PAO1. Heat-inactivated PAO1 was chosen to obtain more reproducible results compared with live P. aeruginosa. We have formally demonstrated that the induction of IL-8 mRNA by PAO1 always occurs, although to a variable extent (31). Figure 6E demonstrates that when IB3–1 cells are

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Figure 3. Predicted secondary structure of the 39-UTR region of IL-8 mRNA based on the UCSC genome browser (genome.ucsc.edu), and of miR-93 and miR-494 by UNAFold Web Server (mfold.rna.albany.edu). Magnification of the central portion of the 39-UTR of the IL-8 mRNA points out the possible interaction between the IL-8 39-UTR target strand and the seed region of the lowest energy miR-93 (A) and miR-494 (B) potential stem loops. (C and D) Evidence for IL-8 as a direct target of miR-93. (C) The miR-93 target sites of the 39-UTR region of IL-8 mRNA are conserved throughout evolution. The results shown have been obtained by the program TargetScan Release 6.2 (www.targetscan.org). (D) Interaction of miR-93 with IL-8 mRNA and other relevant mRNAs already demonstrated as miR-93 target molecules.

infected with heat-inactivated PAO1, the expected increase of IL-8 mRNA occurs (z 15-fold induction; Figure 6E, gray bar). However, if IB3–1 are transfected with

a pre–miR-93 RNA, the level of miR-93 expression increases as expected (Figure 6D, black bar), and a sharp decrease of PAO1-induced IL-8 mRNA occurs

Fabbri, Borgatti, Montagner, et al.: microRNA-93 and IL-8 mRNA in CF

(Figure 6E, black bar). This is confirmed by Bio-plex analysis performed on the cell culture supernatants, in which a sharp decrease of released IL-8 was found in 1149

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Figure 4. Comparison of the effects of PAO1 infection on the expression of miR-93, minichromosome maintenance complex component 7 (MCM7), and IL-8. (A) Analysis of the relative expression of miR-93 (white bars), MCM7 (gray bars), and IL-8 (black bars) after infection with heat-inactivated PAO1. After 24 hours of treatment, RNA was extracted, and RT-qPCR analysis was performed. The data reported show a high-level expression of miR-93 and MCM7, low-level expression of IL-8 in IB3–1 cells, and up-regulation of IL-8 associated with down-regulation of miR-93 and MCM7 in PAO1-infected IB3–1 cells. Data represent the average 6 SD (n = 3). (B, C) Effects of PAO1 infection on key transcription factors. (B) Presence of E2F-1 and MYC transcription factor binding sites within the promoter region of the MCM7 gene. The arrow shows the transcription starting site. (C) Bio-plex analysis of the nuclear content of NF-kB, MYC, and E2F-1 level in IB3–1 cells and IB3–1 cells infected with heat-inactivated PAO1. Infected cells (gray bars) show a higher level of NF-kB and lower levels of MYC and E2F-1 compared with uninfected IB3–1 (black bars). Data represent the average 6 SD (n = 3). Statistical significance reported in A and C is expressed in comparison to uninfected IB3–1 cells.

pre–miR-93/PAO1–exposed IB3–1 cells (Figure 6F). These results support the concept that forced up-regulation of miR93 counteracts the effects mediated by PAO1 infection (i.e., miR-93 downregulation and IL-8 induction). To determine whether the effects of antagomiR-93 and pre–miR-93 are observed in other bronchial cell lines (derived from subjects with and without CF), the 1150

experiments shown in Figure 6G–6N were performed. Also, in CuFi-1 (Figures 6G–6I) and NuLi-1 cells (Figure 6L–6N), we observed that mimicking PAO1 downregulation of miR-93 by using an antagomiR-93 leads to induction of IL-8 (analyzed at the mRNA and protein levels; Figures 6G and 6L and Figures 6H and 6M, respectively), as found in IB3–1 cells (Figures 6B and 6C). In addition, when

pre–miR-93 was transfected to CuFi-1 and NuLi-1 cells before infection with PAO1, a sharp decrease of PAO1-induced IL-8 mRNA occurred (Figures 6G and 6L), in agreement with data obtained using IB3–1 cells (Figure 6E). This was confirmed by Bioplex analysis performed in the media, in which a much lower release of IL-8 was found in pre–miR-93/PAO1–exposed CuFi1 and NuLi-1 cells compared with control cells (Figures 6I and 6N). To verify the possible role of miR-93 in PAO1-mediated induction and to compare the miR-93 dependency of IL-8 gene expression with that displayed by other miR-93–regulated genes, we focused on IL-6 (one of the major PAO1 induced genes in IB3–1 cells) (45) and VEGF-A, a validated miR-93 regulated gene (46). To calculate the miR-93 dependency index (miR-93INDEX), we applied the following algorithm: foldpre-miR-93/foldantagomiR-93. The miR-93INDEX values are shown in Figure E2, which reports a summary of the data from five experiments using IB3–1 cells. The IL-8 gene displays high miR-93 dependency (i.e., low miR-93INDEX: 0.45 6 0.08), similar to that exhibited by VEGF-A (miR-93INDEX: 0.52 6 0.19). Furthermore, IL-6 exhibits low miR-93INDEX (0.51 6 0.17). These data confirm the role of miR93 in regulating IL-8 gene expression, but suggest that miR-93 might be involved in the regulation of other PAO1-induced genes. Further experiments are needed to clarify this important issue.

Discussion In this study we have analyzed by microarray the miR-profile of P. aeruginosa (PAO1)-infected IB3–1 cells, which express, in these experimental conditions, very high levels of cytokines and chemokines, thus mimicking the inflammatory response commonly observed in the lungs of patients with CF (19). The microarray data were confirmed by RT-qPCR analysis and allowed us to identify miR-93 as one of the miRs expressed at the highest levels in IB3–1 cells. When RT-qPCR was performed on PAO1-infected IB3–1 cells, we observed that miR-93 was reduced. IL-8 mRNA (which contains sequences for miR93) is the most expressed among the induced mRNA sequences in PAO1infected IB3–1 cells. On the basis of these observations, we can speculate that down-

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Figure 5. Effect of the treatment with antagomiR-93 or pre–miR-93 on luciferase activity controlled by the IL-8 39-UTR region. (A, B) IB3–1 cells were transfected with 1 mM antagomiR-93 (A) or pre–miR-93 (B) (white bars) and with the IL-8 39-UTR-pMirTarget Vector as described in MATERIALS AND METHODS; for comparison, IB3–1 cells were also transfected with equivalent amounts of negative controls (black bars). The luciferase activity was normalized with red fluorescent protein levels produced by the vector. After 48 hours, IB3–1 cells were lysed, and the luminescence intensity was detected using a luciferase assay kit. All assays were repeated in five independent experiments. (C–E) Effect of the treatment with pre–miR-93 on luciferase activity carried on by the IL-8 39-UTR pMirTarget Vector (C), the same vector deleted of the IL-8 39-UTR region (D) and the same vector containing an IL-8 39-UTR region in which the miR-93 hypothetical sequence was mutagenized (E). Results represent the average 6 SD. MUT, mutagenized.

regulation of miR-93 is associated with upregulation of IL-8 in PAO1-infected IB3–1 cells. Supporting this hypothesis is the fact that the 39-UTR sequences of the IL-8 mRNA, which are potential targets of the miR-93 at the level of the RNA-induced silencing complex, are highly conserved throughout molecular evolution. The down-regulation of miR-93 after PAO1 infection was confirmed in another CF cell line (CuFi-1) and in a non-CF bronchial cell line (NuLi-1).

The experiments performed with a luciferase vector carrying the 39-UTR of the IL-8 mRNA cloned downstream the luciferase sequences fully support this hypothesis because treatment with antagomiR-93 increases luciferase activity, which, conversely, was strongly reduced by a treatment with pre–miR-93. To verify whether the miR-93 sequence plays a major role in this behavior, we have developed a vector carrying a 39-UTR of the IL-8 gene with a mutagenized miR-93 binding site.

Fabbri, Borgatti, Montagner, et al.: microRNA-93 and IL-8 mRNA in CF

No changes of luciferase activity were found in this case after treatment with pre–miR-93, suggesting an important role of miR-93 for the posttranscriptional control of IL-8 gene expression. To further verify this hypothesis, we performed two complementary experiments. In the first experiment, the down-modulation of miR-93 after PAO1 infection was counteracted by pre–miR-93 transfection and IL-8 mRNA and IL-8 secretion analyzed by RT-qPCR and Bioplex assays. In the second experiment, uninfected cells were treated with antagomiR-93 to mimic the PAO1mediated down-regulation of this microRNA, and the levels of IL-8 mRNA and IL-8 secretion were analyzed. This was performed using the IB3–1, CuFi-1, and NuLi-1 cells. The results obtained clearly indicate that when miR-93 content is increased after transfection of PAO1-infected cells with pre–miR-93, IL-8 gene expression is significantly inhibited. In agreement, when antagomiR-93 is transfected to cells, an increase of IL-8 production occurs without PAO1 infection. Altogether, these results support the concept that miR-93 is involved in the posttranscriptional regulation of IL-8 gene expression. Our experiments do not exclude the possibility that miR-93 interacts with other RNA targets involved in proinflammatory phenotype of PAO1-infected cells. An observation suggesting that this issue deserves attention in future experimental work is included in Figure E2. In this experiment, performed on IB3–1 cells, the IL-8 gene was found to exhibit high miR-93 dependency, together with another PAO1-induced genes, IL-6. Our data do not exclude the possibility that other 39-UTR regulatory factors or other microRNAs are involved in the regulation of IL-8 gene expression. Despite the fact that 34 microRNAs are potentially able to recognize the 39-UTR sequences of the human IL-8 mRNA (see Figure 1B), only miR-93 and miR-494 are expressed and down-regulated when a 2-fold limit is chosen to extract the down-regulated microRNAs in the microarray experiments. Further experiments focusing on the other microRNAs will clarify this point. The miR-93 belongs to a cluster of three miRNAs and is located in the 13th intron of the gene of human chromosome 7, MCM7. MicroRNA-93 is cotranscribed with the 1151

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Figure 6. Effects of treatment of IB3–1 cells with antagomiR-93 or pre–miR-93. (A–C) IB3–1 cells were untreated (-) (white boxes), transfected with antagomiR-93 (black boxes), or transfected with a negative control miR sequence (gray boxes). The expression of miR-93 (A), IL-8 mRNA (B), and IL-8 protein secretion (C) was analyzed. (D–F) IB3–1 cells were transfected with pre–miR-93 for 24 hours and infected with PAO1 for 24 hours, and the expression of miR-93 (D), IL-8 mRNA (E), and IL-8 protein secretion (F) was analyzed. (G–N) Effects of treatment of CuFi-1 (G–I) and NuLi-1 (L–N) cells with antagomiR93 or pre–miR-93 plus PAO1 (as done for IB3–1 cells; A–C) on IL-8 mRNA content (G, L) and IL-8 release (H, I, M, and N), as indicated. Analysis of miR-93 and IL-8 mRNA was performed by quantitative RT-PCR, and release of IL-8 was quantified by Bio-plex analysis. White boxes: untreated cells (-); gray boxes: negative control miR sequence; black boxes: cells treated with antagomiR-93 or pre–miR-93 plus PAO1. Data represent the average 6 SD (n = 3). Statistical significance is expressed with respect to IB3–1 cells treated with negative control sequences. *P , 0.05; **P , 0.01.

MCM7 primary transcript. Abnormal expression of miR-93 in many different malignancies has been reported in several studies (36, 39, 47, 48) involving genes associated with cell proliferation, apoptosis, and tissue turnover or function. Our data allow proposing a hypothetical scheme (Figure 7) leading to up-regulation of IL-8 gene expression after P. aeruginosa infection of CF cells. In uninfected cells, low NF-kB recruitment causes a low transcription of the IL-8 gene; on the other hand, high expression of MCM7/miR-93 cluster occurs associated with high 1152

nuclear recruitment of MYC and E2F-1 transcription factors. These two independent molecular pathways concurrently participate in causing low IL-8 expression. In CF-infected cells, NF-kB activation leads to an increased transcription of the IL-8 gene; at the same time, a decrease of MYC and E2F-1 leads to (1) a decrease of a transcription of MCM7/miR-93 cluster, (2) a decrease of miR-93 accumulation, and (3) a further increase of IL-8 gene expression. P. aeruginosa infection has dramatic differential effects on transcription factors:

NF-kB increases, whereas the content of MYC and E2F-1 decreases, as the two TFs involved in the regulation of the MCM7 gene, well in agreement with the downregulation of MCM7 mRNA and miR-93. The content of other TFs (YY-1, GATA-1) recognizing the MCM7 promoter decreases (data not shown). To our knowledge, this is the first observation of a possible link between miR-93 expression and IL-8 induction in P. aeruginosa–infected CF cells. The results reported in the present paper, therefore, suggest that, in addition to transcriptional

American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 6 | June 2014

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Figure 7. Model for regulation of IL-8 gene expression in cystic fibrosis (CF) cells. (A) High levels of MYC and E2F-1 transcription factors lead to the expression of high constitutive levels of miR-93 originating from the 13th intron of MCM7 gene. At the same time, low recruitment of the NF-kB to the IL-8 gene promoter (33) leads to low levels of IL-8 mRNA transcripts. These two combined pathways are concurrent in originating low levels of IL-8 production. (B) CF cells infected with PAO1. As previously demonstrated (33), in these cells high levels of NF-kB transcription factor are preferentially recruited on the IL-8 gene promoter (leading to high expression of the IL-8 mRNA). At the same time, lower levels of MYC and E2F-1 transcription factors reduce miR-93 transcription, causing lower miR-93/IL-8 interactions. These two combined pathways are concurrent in originating high levels of IL-8 production in P. aeruginosa–infected cells.

NF-kB–dependent up-regulation of IL-8 gene expression, IL-8 protein secretion might depend on interactions of the IL-8 mRNA with inhibitory microRNAs, including miR-93. This observation should be considered together with other reports on the relationship between CF, IL-8 gene expression, and microRNAs. In this respect, Bhattacharyya and colleagues screened a miRNA library for differential expression in DF508-CFTR and WT CFTR lung epithelial cell lines and found that expression of miR-155 was more than 5fold elevated in CF IB3–1 lung epithelial cells in culture, compared with control IB3–1/S9 cells (49). Clinically, miR-155 was also highly expressed in CF lung epithelial

cells and circulating CF neutrophils collected from patients with CF. In this case, the up-regulation of miR-155 specifically reduced levels of SHIP1, thereby promoting PI3K/Akt activation, and production of IL-8. In our P. aeruginosa induced system, down-regulation of miR93 directly leads to a stabilization of the IL-8 mRNA. In addition to basic science implications, our data might be of interest in applied biomedicine because we demonstrate that forced expression of miR-93 is able to reduce IL-8 gene expression; therefore, molecules mimicking pre–miR-93 activity might reduce IL-8–dependent inflammatory

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