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Article

In Vitro Identification of New Transcriptomic and miRNomic Profiles Associated with Pulmonary Fibrosis Induced by High Doses Everolimus: Looking for New Pathogenetic Markers and Therapeutic Targets

by
Simona Granata
1,
Gloria Santoro
1,
Valentina Masola
1,
Paola Tomei
1,
Fabio Sallustio
2,3,
Paola Pontrelli
2,
Matteo Accetturo
2,
Nadia Antonucci
1,
Pierluigi Carratù
4,
Antonio Lupo
1 and
Gianluigi Zaza
1,*
1
Renal Unit, Department of Medicine, University of Verona, Piazzale Stefani 1, 37126 Verona, Italy
2
Department of Emergency and Organ Transplantation, University of Bari “Aldo Moro”, Piazza Giulio Cesare 11, 70124 Bari, Italy
3
Department of Basic Medical Sciences, Neuroscience and Sense Organs, University of Bari “Aldo Moro”, Piazza Giulio Cesare 11, 70124 Bari, Italy
4
Department of Respiratory Diseases, University of Bari “Aldo Moro”, Piazza Giulio Cesare 11, 70124 Bari, Italy
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(4), 1250; https://doi.org/10.3390/ijms19041250
Submission received: 23 March 2018 / Revised: 17 April 2018 / Accepted: 17 April 2018 / Published: 20 April 2018
(This article belongs to the Special Issue mTOR in Human Diseases)
Browse Figures
Versions Notes

Abstract

:
The administration of Everolimus (EVE), a mTOR inhibitor used in transplantation and cancer, is often associated with adverse effects including pulmonary fibrosis. Although the underlying mechanism is not fully clarified, this condition could be in part caused by epithelial to mesenchymal transition (EMT) of airway cells. To improve our knowledge, primary bronchial epithelial cells (BE63/3) were treated with EVE (5 and 100 nM) for 24 h. EMT markers (α-SMA, vimentin, fibronectin) were measured by RT-PCR. Transepithelial resistance was measured by Millicell-ERS ohmmeter. mRNA and microRNA profiling were performed by Illumina and Agilent kit, respectively. Only high dose EVE increased EMT markers and reduced the transepithelial resistance of BE63/3. Bioinformatics showed 125 de-regulated genes that, according to enrichment analysis, were implicated in collagen synthesis/metabolism. Connective tissue growth factor (CTGF) was one of the higher up-regulated mRNA. Five nM EVE was ineffective on the pro-fibrotic machinery. Additionally, 3 miRNAs resulted hyper-expressed after 100 nM EVE and able to regulate 31 of the genes selected by the transcriptomic analysis (including CTGF). RT-PCR and western blot for MMP12 and CTGF validated high-throughput results. Our results revealed a complex biological network implicated in EVE-related pulmonary fibrosis and underlined new potential disease biomarkers and therapeutic targets.

1. Introduction

Everolimus (EVE), marketed as Certican, is a pharmacological agent widely used in the anti-rejection therapy of solid organ transplantation and in the treatment of certain tumors (e.g., in advanced renal cell carcinoma, subependymal giant cell astrocytoma associated with tuberous sclerosis, pancreatic neuroendocrine tumors, breast cancer) [1]. Similar to Sirolimus and Tamsilorimus, it exerts its immunosuppressive activity by inhibiting mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase-related protein that controls cell cycle, protein synthesis, angiogenesis and autophagy [2]. These important multi-factorial biological/cellular effects allow this drug to avoid/minimize the onset of acute rejection episodes and to slow down the progression of chronic allograft lesions [3,4].
However, some authors have reported a high rate of discontinuation secondary to side effects after the introduction of this drug [5,6,7]. Among them, pneumonitis or interstitial lung disease with a range of pulmonary histopathologic changes (including alveolar hemorrhage, pulmonary alveolar proteinosis, focal fibrosis, bronchiolitis obliterans organizing pneumonia) have been largely reported in clinical records and they have been associated with worsened patients’ clinical outcomes and drug discontinuation [8,9,10,11,12,13,14,15,16]. The incidence of this complications is 2–11%, frequently reported between 1 and 51 months after the beginning of mTOR inhibitor therapy [17,18,19].
The pathogenic mechanism underlying lung toxicity is multi-factorial and epithelial to mesenchymal transition (EMT) of airway cells seems to have a pivotal role [20,21,22,23]. Our group has recently demonstrated that high doses of EVE are associated with a reprogramming of gene expression in several epithelial cell lines (airway, renal epithelial proximal tubular and hepatic cells) with a consequent loss of their phenotype (junctions and apical-basal polarity) and the acquisition of mesenchymal traits increasing the motility and enabling the development of an invasive and pro-fibrotic phenotype [24,25,26].
High dosage of EVE eliminating negative crosstalk from mTORC1/S6K, leads to activation of mTORC2 that enhances AKT phosphorylation at Ser473 and stimulates PI3K-AKT signaling that induces renal fibrosis [26,27,28,29,30].
The pro-fibrotic attitude of EVE has also been confirmed in vivo in renal transplant patients through the estimation of an arbitrary pulmonary fibrosis index score in renal transplant patients chronically treated with this drug. In this patients’ subset, high blood trough level of EVE was associated with a high rate of pulmonary signs of fibrosis [24].
However, although the aforementioned studies and the large clinical evidences, the complete biological machinery involved in this condition has not been completely clarified.
Therefore, we employed, for the first time, a highthroughput approach combining a transcriptomic with a miRNome analysis to study the capability of EVE to induce pro-fibrotic changes in primary bronchial epithelial cells.
All together our results could represent a step forward in the comprehension of the mTOR-I associated biological machinery and in the identification of new targets for therapeutic interventions.

2. Results

2.1. High Dosage Everolimus (EVE) Induced Epithelial to Mesenchymal Transition (EMT) of BE63/3 (Primary Bronchial Epithelial Cells)

To confirm our previous results obtained in immortalized bronchial and pulmonary cell lines [24], we decided to measure by Real Time-PCR the expression level of alpha smooth muscle actin (α-SMA), vimentin (VIM), and fibronectin (FN) in BE63/3 treated for 24 h with 2 different dosages of EVE (5 and 100 nM) chosen according to literature evidences [31,32,33,34] and previous experiments performed by our research group in different cell lines [24,25,26].
Only high dose of EVE (100 nM), similarly to TGF-β (20 ng/mL), increased the mRNA level of the EMT-related markers (Figure 1A–C). Moreover E-cadherin resulted downregulated although it did not reach a statistically significant level (Figure S1). Contrarily, 5 nM EVE was ineffective (Figure 1A–C).
Additionally, high dosage of EVE was also able to reduce the transepithelial resistance (TER) evaluated by a Millicell-ERS ohmmeter indicating dysfunctional tight junctions (Figure 1D).

2.2. Transcriptomic Analysis Revealed That High Dosage of EVE Up-Regulated Genes Involved in Collagen Synthesis and Metabolism

Gene expression profiling evaluated by transcriptomic analysis revealed that in vitro treatment of BE63/3 cells with 100 nM EVE for 24 h deregulated 147 probe sets (corresponding to 125 genes): 60/147 probe sets (47 genes) resulted up-regulated while 87/147 probe sets (corresponding to 78 genes) were down-regulated (≥1.5-fold change) in EVE-treated cells compared with control (CTR) (Table 1). According to enrichment analysis, selected genes belonged to 44 pathways (Table 2) and 5 of them were involved in collagen synthesis/metabolism and regulation of stress fiber assembly. Interestingly, connective tissue growth factor (CTGF) was a representative gene in all these pro-fibrotic pathways.
Instead, low dosage EVE (5 nM) was able to change the expression level of only 33 probe sets (24 genes): 25/33 probe sets (20 genes) were hyper-expressed and 4 probe sets (4 genes) down-regulated after treatment (Table 3). None of the selected pathways was associated with the pro-fibrotic cellular machinery (Table 4).
Principal component analysis (PCA) and volcano plot showed the degree of separation of untreated versus treated cells at both EVE dosages (Figure 2).

2.3. MiRNome Analysis Identified Specific MicroRNAs Deregulated by EVE

To gain insights into the mechanism leading to EMT induced by EVE and to discover possible regulatory miRNAs of this effect, we performed a miRNome analysis by miRNA Complete Labeling and Hybridization kit. Statistical analysis identified three miRNAs up-regulated after high dosage (100 nM) (Table 5) and four after treatment with EVE at low dosage (5 nM) (Table 6). Among these, miR-8485 was the most up-regulated miRNA (more than 4-fold changes in both treatments).
By matching mRNA and miRNA expression data, we found that 31 genes were specific target of the three identified miRNAs (Table 7).

2.4. Gene Expression and Protein Analysis for Matrix Metalloproteinase 12 (MMP12) and Connective Tissue Growth Factor (CTGF) Validated High-Throughput Results

In order to validate microarray results, we measured by Real-Time PCR the level of mRNA expression of MMP12 and CTGF. Both transcripts were up-regulated after treatment with 100 nM EVE. Contrarily 5 nM EVE had no effect (Figure 3A,B). In addition, western blot analysis of CTGF confirmed gene expression results at protein level (Figure 3C,D).

2.5. Validation of Transcriptomic Results in an Additional Primary Cell Line (BE121/3)

To confirm transcriptomic results, we decided to measure the expression level of 8 selected genes (involved in EMT) up-regulated after high dosage EVE in a new primary bronchial epithelial cell line. As showed in Figure 4, results were in line with those obtained in BE63/3 (Figure 4).

2.6. High Dosage EVE Up-Regulated CTGF and Collagen1 in Fibroblasts and Hepatic Stellate Cells

To validate the pro-fibrotic effect of high dosage EVE we measured the expression level of collagen1 and CTGF in NIH/3T3 (mouse embryo fibroblast cell line) treated with EVE.
Interestingly, also in fibroblasts high dosage EVE up-regulated the protein levels of collagen1 and CTGF (Figure 5).
Also, in hepatic stellate cells high dosage EVE induced the up-regulation of CTGF and collagen1 (Figure S2).

3. Discussion

Pulmonary fibrosis is a potential serious adverse effect following administration of mTOR-I in patients undergoing solid organ transplantation or receiving anti-cancer therapies. It is generally accepted that pulmonary disease is related to mTOR-I therapy, whether the following conditions are present: (1). The symptoms of pulmonary disease occur after initiation of mTOR-I therapy; (2). Infection, other pulmonary diseases or toxicity associated with other drugs are excluded; (3). mTOR-I minimization or discontinuation lead to resolution of the symptoms. In fact, the dose-dependent effect was proved by the observation of this disease particularly in patients receiving high doses of mTOR-I.
Pulmonary manifestations in these patients are numerous and include several clinical/histological phenotypes (e.g., focal pulmonary fibrosis, bronchiolitis obliterans with organizing pneumonia) [8,9,35,36].
This multi-factorial and heterogeneous clinical condition is often responsible for drug discontinuation and it requires long and expensive clinical evaluations and treatments (e.g., antibiotics, corticosteroids, immunosuppressive drugs) [14] with the involvement of a multidisciplinary team of experts (e.g., pulmonologists, infectivologists, nephrologists).
The etiopathogenic mechanism of pulmonary toxicity associated with mTOR-I therapy is not known and several in vivo and in vitro studies have tried to define the underlying mechanisms. It has been proposed a T cell-mediated autoimmune response induced when pulmonary cryptic antigens are exposed, leading to lymphocytic alveolitis and interstitial pneumonitis [15]. Other possible pathogenic mechanisms could be a delayed-type hypersensitivity reaction [9] or pulmonary inflammation as a direct effect of mTOR-I to stimulate cells of the innate immune system to produce proinflammatory cytokines [37,38].
Additionally, Ussavarungsi et al. have reported that sirolimus may induce granulomatous interstitial inflammation and proposed a mechanism of T-cell mediated hypersensitivity reaction triggered by circulating antigens or immune complexes in the lungs [39].
Moreover, several authors have emphasized the pathogenetic role of the EMT of bronchial epithelial cells in these important Everolimus (EVE)-related adverse events [20,21,22,23].
To obtain more insights, we decided to employ, for the first time, innovative high throughput technologies, to identify new elements involved in the biological/cellular reprogramming induced by high dose of mTOR-I and leading to fibrosis.
In vitro experiments using classical bio-molecular strategies, confirmed, in primary bronchial epithelial cell lines, our previous results demonstrating the ability of high dosages EVE to induce EMT. In particular, 100 nM EVE caused the up-regulation of EMT-related genes (α-SMA, VIM, FN) and reduced the trans-epithelial resistance to the same levels induced by TGF-β. Then, high doses of this drug significantly changed the expression level of 125 genes (47 up- and 78 down-regulated).
Several of the selected genes were target of miR-8485, the top significant and up-regulated microRNA (miRNA) by EVE 100 nM. Other 2 miRNAs were identified after the same treatment: miR-937-5p and miR-5194. Except for miR-8485, at our knowledge, none of them has been previously associated with fibrosis or supposed to be regulatory of genes implicated in this process. It’s unquestionable that further studies are warranted to confirm the involvement of these miRNAs in EVE induced EMT since all identified miRNAs were up-regulated demonstrating their possible role as enhancer of fibrotic machinery. This could be in line with recent findings suggesting that miRNA-mediated down-regulation is not a one-way process and some miRNAs could up-regulate gene expression in specific cell types and conditions with distinct transcripts and proteins [40,41]. It is noteworthy that these miRNAs are up-regulated also after treatment with 5 nM EVE. Many reasons could be responsible of this effect. In particular, the expression of these miRNAs could be regulated by several factors and networks (some of them also unrelated to mTOR-I treatment). Additional studies are needed to clarify the role of miRNA in EVE-mediated pro-fibrotic effect.
Moreover, analyzing the results of the transcriptomic analysis and the hypothetic targets of miR-8485, we found that connective tissue growth factor (CTGF), a protein secreted into the extracellular environment where it interacts with distinct cell surface receptors, growth factors and extra-cellular matrix [42,43] was one of the top scored genes. Gene expression by RT-PCR and protein analysis by western blotting confirmed the result obtained by microarray.
It is well known that CTGF modulates the activities of TGF-β or vascular endothelial growth factor (VEGF), with consequent pro-fibrotic and angiogenetic effects [44,45,46,47]. However, the overexpression of CTGF in fibroblast of mice caused tissue fibrosis in vivo [48] without involving the canonical TGF-β pathway. This is in line with several reports that demonstrated a mTOR-I dose-related induction of CTGF at gene and protein levels in vitro and in vivo [49,50,51,52].
Moreover, Xu et al. have demonstrated that rapamycin, an analogue of EVE, exerted a profibrotic effect in lung epithelial cells as well as in lung fibroblasts via up-regulation of CTGF expression and PI3K/AKT pathway [50,51]. Similarly, Mikaelian et al. using a combination of RNAi and pharmacological approaches showed that inhibition of mTOR triggers EMT in mammalian epithelial cells by a mechanism TGF-β independent [53]. In the transplant context it has been described a synergistic fibrotic effect of sirolimus with cyclosporine in kidney also mediated by the up-regulation of CTGF [54,55].
Another interested gene up-regulated by EVE, selected by microarray and validated by RT-PCR, was metalloproteinase 12 (MMP12), a member of the zinc-dependent endopeptidases family able to proteolyze all components of the extracellular matrix [56,57] by degrading collagen, other extracellular filaments, cytokines, growth factors and their receptors. MMP12 has a pivotal role in TGF-β mediated pulmonary fibrosis [58,59].
Interestingly, other identified genes by transcriptomic analysis and target of miR-8485 (Table 7) were Kallmann syndrome-1 gene (KAL1, fold change: 1.705), Limb-bud and heart (LBH, fold change: 1.808) and insulin receptor substrates 2 (IRS2, fold change: 1.646) that resulted up-regulated after 100 nM EVE treatment and Protocadherin 7 (PCDH7, fold change: −1.625) down-regulated by similar treatment. All of them have been described in literature as directly or indirectly involved in the EMT.
KAL1, codes for anosmin-1, a cell adhesion protein in extracellular matrix induced by TGF-β [60,61]. IRS2 expression appears to repress the expression of E-cadherin [62], marker of epithelial cells deregulated during EMT.
LBH is a transcription cofactor with both transcriptional activator and corepressor functions. LBH is a direct Wnt/β-catenin target gene and is induced by TGF-β [63,64]. Wnt/β-catenin signaling activation occurs in cells during EMT [65] and treated with mTOR-I.
Protocadherin 7 is an integral membrane protein having a role in cell–cell recognition and adhesion. Down-regulation of PCDH7 gene was correlated with E-cadherin inhibition [66].
All these findings, although speculatively interesting, need to be validated in vivo. Our study is an hypothesis generating study that should be considered a starting point for bio-molecular study involving transplanted patients or animal models.
Nevertheless, after 21 days in culture, most of the cells were not ciliated and we cannot exclude that differentiation state may have affected the response to EVE (Figure S3).
However, our results suggested that high concentrations of EVE, through the activation of a multi-factorial biological/cellular machinery, may lead to pulmonary fibrosis and underlined potential pathogenetic, diagnostic biomarkers and targets for future pharmacological interventions to introduce in the “day by day” clinical practice. Finally, at a clinical point of view, we confirm that, whenever possible, the dose of EVE should be the minimized in patients with early signs of lung toxicity.

4. Materials and Methods

4.1. Cell Culture Treatment

Primary wild-type bronchial epithelial cells (BE63/3 and BE121/3) were obtained from “Servizio Colture Primarie” of the Italian Cystic Fibrosis Research Foundation (ICFRF) and cultured following the supplier instructions [67]. The protocols to isolate, culture, store, and study bronchial epithelial cells from patients undergoing lung transplant was approved by the Ethical Committee of Gaslini Institute (ethical approval number IGG:192 date of approval: 9/24/2010) under the supervision of the Italian Ministry of Health. Cells were grown on rat tail collagen-coated tissue culture plates in serum-free LHC9/RPMI 1640 medium at 37 °C and 5% CO2.
After 4–5 passages, cells were seeded on Transwell porous inserts. After 24 h from seeding, the medium was switched to DMEM/F12 supplemented with 2% Ultroser G, 2 mM l-glutammine, 100 U/mL penicillin, 100 μg/mL streptomycin.
Exchange of culture medium is repeated every day on both sides of permeable supports up to 5 days. Then the apical culture medium was removed, and the medium was added only in the basolateral side (air-liquid interface) favoring a differentiation of the epithelium (Figure S3). After 11 days the epithelium was treated with EVE (5 nM and 100 nM) and TGF-β (20 ng/mL), an EMT inducer, for 24 h. “The timing of cell culture for gene expression and western blot experiments (17 days) was based on clear instructions supplied by the “Servizio Colture Primarie” of the ICFRF in order to reach the differentiation of epithelium”. Although the in vitro model cannot completely represent the in vivo pharmacokinetic/effect of this drug, we can postulate that 5 nM EVE corresponds to a trough level of approximately 5 ng/mL (drug level frequently reached in the immunosuppressive maintenance therapy of solid organ transplantation), while 100 nM may correspond to very high dosages (trough level more than 50 ng/mL) that patients could reach in anticancer therapy.
NIH/3T3 fibroblasts, purchased from American Type Culture Collection (Manassas, VA, USA) were maintained at 37 °C in DMEM supplemented with 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine. Cells were treated with or without 5 and 100 nM Everolimus for 24 h.

4.2. RNA Extraction and Gene Expression Profiling

Trizol reagent (Invitrogen) was used to extract total RNA and then, yield and purity were checked using a Nanodrop spectrophotometer.
Gene expression data were produced using the HumanHT-12 v3 Expression BeadChip (Release 38, Illumina, San Diego, CA, USA). Five hundred ng total RNA from BE63/3 was used to synthesize biotin-labeled cRNA using the Illumina®TotalPrep™ RNA amplification kit (Applied Biosystems, Foster City, CA, USA). Quality of labelled cRNA was assessed by NanoDrop® ND-100 spectrophotometer and the Agilent 2100 Bioanalyzer. Then, 750 ng biotinylated cRNA was used for hybridization to illumina microarrays that were then scanned with the HiScanSQ.

4.3. Pathway Analysis

The Ingenuity Pathway Analysis software (IPA, Ingenuity System, Redwood City, CA, USA) was used to assess biological relationships among differentially regulated genes. The reference gene selection was performed by own software written in Java program language. The canonical pathways generated by IPA are the most significant for the uploaded data set. Fischer’s exact test with false discovery rate (FDR) option was used to calculate the significance of the canonical pathway.

4.4. MicroRNA Expression Profiling

Fluorescently-labeled miRNAs were generated using the miRNA Complete Labeling and Hybridization kit (Agilent Technologies, Santa Clara, CA, USA), with a sample input of 100 ng of total RNA from BE63/3 and hybridized for 20 h at 55 °C on the Agilent 8 × 60 K Human miRNA Microarray slide (Agilent Technologies), based on miRBase database (Release 21.0). Following hybridization, the slides were washed and scanned using the High-Resolution Microarray C Scanner (Agilent Technologies). The image files were processed using the Agilent Feature Extraction software (v10.7.3): the microarray grid was correctly placed; inlier pixels were identified, and outlier pixels were rejected.

4.5. Real-Time PCR

Five hundred ng total RNA from each sample was reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR amplification reactions were performed in duplicate via SYBR Green chemistry on CFX-connect (Bio-Rad, Hercules, CA, USA) and SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad). Primers for α-SMA, VIM, FN, MMP12, CTGF, CDH6, COL12A1, FAP, KAL1, LBH, PIM1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Qiagen (QuantiTect Primer Assay, Hilden, Germany).
The comparative Ct method (ΔΔCt) was used to quantify gene expression and the relative quantification was calculated as 2−ΔΔCt. Melting curve analysis was employed to exclude non-specific amplification products.

4.6. Western Blot

Equal amounts of proteins were resolved in 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. Non-specific binding was blocked for 1 h at room temperature with non-fat milk (5%) in TBST buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). Membranes were exposed to primary antibodies directed against GAPDH (Santa Cruz sc-25778), CTGF (NovusBio, Littleton, CO, USA) and collagen1 (ORIGENE TA309096) (overnight at 4 °C) and incubated with a secondary peroxidase-conjugated antibody for 1 h at room temperature. The signal was detected with SuperSignals West Pico Chemiluminescent substrate solution (Pierce) according to the manufacturer’s instructions.

4.7. Transepithelial Resistance (TER)

Millicell-ERS ohmmeter with electrodes (Millipore) was used to measure TER (alternating current applied between the electrodes: ±20 μA and frequency: 12.5 Hz). The resistance of the monolayer multiplied by the effective surface area was used to obtain the electrical resistance of the monolayer (Ω cm2). Once stable resistances were obtained, different culture media (control, EVE 5 nM, EVE 100 nM, TGF-β 20 ng/mL) were tested. After the addition of test solutions, measurements were taken at 24 h.

4.8. Statistical Analysis

For transcriptomics statistical analyses were carried out by Genespring GX 11.0 software (Agilent Technologies). Gene probe sets were filtered based on the FDR method of Benjamini–Hochberg and fold-change. Only genes that were significantly (adjusted-p value < 0.05 and fold-change > 1.5) modulated were considered for further analysis.
In the miRNome analysis, after normalization (Quantile method), unpaired t-test (p-value cut-off: 0.05 and fold-change cut-off: 2.0, after Benjamini–Hochberg multiple testing correction) was employed to identify most differentially expressed probes.
For the statistical analysis of RT-PCR and western-blot, differences between control and treated cell were compared using Student’s t-test. A p-value < 0.05 was set as statistically significant.

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/1422-0067/19/4/1250/s1.

Acknowledgments

This study was funded by grants from the Italian Cystic Fibrosis (CF) Research Foundation (FFC#28/2014, Delegazione FFC di Torino, Lodi/Latina, Italy) and from the Fondazione Cariverona 2015. This study was performed in the LURM (Laboratorio Universitario di Ricerca Medica) Research Center, University of Verona, Verona, Italy.

Author Contributions

Gianluigi Zaza, Simona Granata, Valentina Masola conceived and designed the experiments; Simona Granata, Valentina Masola, Gloria Santoro, Nadia Antonucci, Fabio Sallustio, Paola Pontrelli, Matteo Accetturo, Paola Tomei performed the experiments; Gianluigi Zaza, Simona Granata, Antonio Lupo, Pierluigi Carratù analyzed the data; Gianluigi Zaza and Simona Granata wrote the manuscript. All co-authors revised and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Gene expression of epithelial to mesenchymal transition (EMT) related markers. Relative (A) alpha smooth muscle actin (α-SMA), (B) fibronectin (FN) and (C) vimentin (VIM) expression evaluated by Real-time PCR in BE 63/3 cells treated or untreated with Everolimus (EVE) (5 and 100 nM) or TGF-β (20 ng/mL); expression values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mean ± S.D. (error bars) of three separate experiments performed in triplicate. * p < 0.05, ** p < 0.01 vs. control (CTR). (D) Histogram represents transepithelial resistance as percentage change with respect to control cells. * p < 0.05 vs. CTR.
Figure 1. Gene expression of epithelial to mesenchymal transition (EMT) related markers. Relative (A) alpha smooth muscle actin (α-SMA), (B) fibronectin (FN) and (C) vimentin (VIM) expression evaluated by Real-time PCR in BE 63/3 cells treated or untreated with Everolimus (EVE) (5 and 100 nM) or TGF-β (20 ng/mL); expression values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mean ± S.D. (error bars) of three separate experiments performed in triplicate. * p < 0.05, ** p < 0.01 vs. control (CTR). (D) Histogram represents transepithelial resistance as percentage change with respect to control cells. * p < 0.05 vs. CTR.
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Figure 2. Principal Component Analysis (PCA) and Volcano Plot discriminating BE63/3 CTR from EVE treated cells. PCA plots were built using the expression level of all differentially expressed genes obtained from mRNA expression profiling after treatment with (A) 5 nM and (C) 100 nM EVE. Volcano Plot based on fold change (Log2) and p value (−Log10) of all genes identified in BE63/3 after treatment with (B) 5 nM and (D) 100 nM EVE. In both graphs red circles indicate the genes that showed statistically significant change.
Figure 2. Principal Component Analysis (PCA) and Volcano Plot discriminating BE63/3 CTR from EVE treated cells. PCA plots were built using the expression level of all differentially expressed genes obtained from mRNA expression profiling after treatment with (A) 5 nM and (C) 100 nM EVE. Volcano Plot based on fold change (Log2) and p value (−Log10) of all genes identified in BE63/3 after treatment with (B) 5 nM and (D) 100 nM EVE. In both graphs red circles indicate the genes that showed statistically significant change.
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Figure 3. Gene expression of MMP12 and connective tissue growth factor (CTGF). mRNA level of (A) MMP12 and (B) CTGF evaluated by real-time PCR in BE63/3 cells treated or not with EVE (5 and 100 nM). Data were normalized to GAPDH expression. Mean ± SD (error bars) of two separate experiments performed in triplicate. ** p < 0.001, * p < 0.05 vs. CTR. (C) Representative western blotting experiments for CTGF. (D) Histogram represents the mean ± SD of CTGF protein level. GAPDH was included as loading control. ** p < 0.001 vs. CTR.
Figure 3. Gene expression of MMP12 and connective tissue growth factor (CTGF). mRNA level of (A) MMP12 and (B) CTGF evaluated by real-time PCR in BE63/3 cells treated or not with EVE (5 and 100 nM). Data were normalized to GAPDH expression. Mean ± SD (error bars) of two separate experiments performed in triplicate. ** p < 0.001, * p < 0.05 vs. CTR. (C) Representative western blotting experiments for CTGF. (D) Histogram represents the mean ± SD of CTGF protein level. GAPDH was included as loading control. ** p < 0.001 vs. CTR.
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Figure 4. Gene expression in BE121/3. mRNA level of (A) CDH6, (B) COL12A1, (C) CTGF, (D) FAP, (E) KAL1, (F) LBH, (G) MMP12, (H) PIM1 evaluated by real-time PCR in BE121/3 cells treated or not with EVE (5 and 100 nM). Data were normalized to GAPDH expression. Mean ± SD (error bars) of two separate experiments performed in triplicate. ** p < 0.001, * p < 0.05 vs. CTR.
Figure 4. Gene expression in BE121/3. mRNA level of (A) CDH6, (B) COL12A1, (C) CTGF, (D) FAP, (E) KAL1, (F) LBH, (G) MMP12, (H) PIM1 evaluated by real-time PCR in BE121/3 cells treated or not with EVE (5 and 100 nM). Data were normalized to GAPDH expression. Mean ± SD (error bars) of two separate experiments performed in triplicate. ** p < 0.001, * p < 0.05 vs. CTR.
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Figure 5. Protein levels of collagen1 and CTGF in NIH/3T3 cells. (A) Representative western blotting experiments for collagen1 and CTGF. Histograms represent the mean ± SD of (B) collagen1 and (C) CTGF protein levels. GAPDH was included as loading control. ** p < 0.001, * p < 0.05 vs. CTR.
Figure 5. Protein levels of collagen1 and CTGF in NIH/3T3 cells. (A) Representative western blotting experiments for collagen1 and CTGF. Histograms represent the mean ± SD of (B) collagen1 and (C) CTGF protein levels. GAPDH was included as loading control. ** p < 0.001, * p < 0.05 vs. CTR.
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Table
Table 1. List of the differentially expressed probe sets after treatment with 100 nM EVE.
Table 1. List of the differentially expressed probe sets after treatment with 100 nM EVE.
Probe IDFold ChangeRegulationSymbolEntrez Gene IDDefinition
47606262.275UpMMP124321matrix metallopeptidase 12 (macrophage elastase), mRNA.
47802092.218UpMMP124321matrix metallopeptidase 12 (macrophage elastase) mRNA.
6700411.925UpAKAP129590A kinase (PRKA) anchor protein (gravin) 12, transcript variant 2, mRNA.
67707461.903UpLOC728715728715similar to hCG38149 (LOC728715), mRNA.
46400861.814UpFOXQ194234forkhead box Q1, mRNA.
28102461.808UpLBH81606limb bud and heart development homolog (mouse) (LBH), mRNA.
63302701.804UpGPC42239glypican 4, mRNA.
66202011.789UpKLHL2454800kelch-like 24 (Drosophila), mRNA.
56906871.783UpCTGF1490connective tissue growth factor, mRNA.
54205771.775UpCLCA422802chloride channel, calcium activated, family member 4, mRNA.
26402921.769UpCTGF1490connective tissue growth factor, mRNA.
10704771.753UpALDH1A1216aldehyde dehydrogenase 1 family, member A1, mRNA.
31303011.729UpPIM15292pim-1 oncogene, mRNA.
66200081.705upKAL13730Kallmann syndrome 1 sequence, mRNA.
40405761.704upIL63569interleukin 6 (interferon, beta 2), mRNA.
18203151.677upC4orf26152816chromosome 4 open reading frame 26 (C4orf26), mRNA.
19901421.671upC20orf11492747chromosome 20 open reading frame 114 (C20orf114), mRNA.
19406471.668upHBP126959HMG-box transcription factor 1, mRNA.
26403241.665upSLC46A3283537solute carrier family 46, member 3, mRNA.
38002411.651upCDH61004cadherin 6, type 2, K-cadherin (fetal kidney), mRNA.
61107361.646upIRS28660insulin receptor substrate 2, mRNA.
46100561.641upFLRT223768fibronectin leucine rich transmembrane protein 2, mRNA.
64206871.638upPLUNC51297palate, lung and nasal epithelium carcinoma associated, transcript variant 2, mRNA.
64204651.625upGABARAPL123710GABA(A) receptor-associated protein like 1, mRNA.
47801281.625upATF3467activating transcription factor 3, transcript variant 4, mRNA.
1602421.622upC13orf1528984chromosome 13 open reading frame 15 (C13orf15), mRNA.
26507091.620upCDH111009cadherin 11, type 2, OB-cadherin (osteoblast), mRNA.
22307671.615upLOC387825387825misc_RNA (LOC387825), miscRNA.
68602281.610upC5orf41153222chromosome 5 open reading frame 41 (C5orf41), mRNA.
65107541.609upALDH1A1216aldehyde dehydrogenase 1 family, member A1, mRNA.
19802551.605upRNF3980352ring finger protein 39, transcript variant 2, mRNA.
68404911.604upC5orf41153222chromosome 5 open reading frame 41 (C5orf41), mRNA.
42802281.595upIVNS1ABP10625influenza virus NS1A binding protein, mRNA.
50800211.593upBIRC3330baculoviral IAP repeat-containing 3, transcript variant 1, mRNA.
64001311.589upCYP24A11591cytochrome P450, family 24, subfamily A, polypeptide 1, nuclear gene encoding mitochondrial protein, mRNA.
71602391.580upFOSB2354FBJ murine osteosarcoma viral oncogene homolog B, mRNA.
3806891.578upTSC22D18848TSC22 domain family, member 1, transcript variant 1, mRNA.
30600951.574upCOL12A11303collagen, type XII, alpha 1, transcript variant short, mRNA.
14102091.571upSGK16446serum/glucocorticoid regulated kinase 1, transcript variant 1, mRNA.
21905531.556upFZD68323frizzled homolog 6 (Drosophila), mRNA.
45700751.544upKIAA164157730KIAA1641, transcript variant 7, mRNA.
50906261.540upFAP2191fibroblast activation protein, alpha, mRNA.
66205381.540upUBL35412ubiquitin-like 3, mRNA.
59603981.537upNT5E49075′-nucleotidase, ecto (CD73), mRNA.
55707311.533upC8orf456892chromosome 8 open reading frame 4 (C8orf4), mRNA.
8306391.531upLOC653778653778similar to solute carrier family 25, member 37 (LOC653778), mRNA.
32901871.529upPCMTD1115294protein-l-isoaspartate (d-aspartate) O-methyltransferase domain containing 1 (PCMTD1), mRNA.
34406701.517upLOC402251402251similar to eukaryotic translation elongation factor 1 alpha 2 (LOC402251), mRNA.
6303151.514upDHRS910170dehydrogenase/reductase (SDR family) member 9, transcript variant 1, mRNA.
14101611.513upKLHL551088kelch-like 5 (Drosophila), transcript variant 3, mRNA.
41505751.513upLETMD125875LETM1 domain containing 1, transcript variant 2, mRNA.
72104971.513upNUAK19891NUAK family, SNF1-like kinase, 1, mRNA.
12404401.511upTXNIP10628thioredoxin interacting protein, mRNA.
47607471.509upTPST18460tyrosylprotein sulfotransferase 1, mRNA.
23602201.508upMATR39782matrin 3, transcript variant 1, mRNA.
38004311.508upRCOR355758REST corepressor 3, mRNA.
43904501.504upSGK6446serum/glucocorticoid regulated kinase, mRNA.
24504651.503upCYBRD179901cytochrome b reductase 1, mRNA.
61100531.501upZNF327580zinc finger protein 32, transcript variant 2, mRNA.
45703981.501upF2R2149coagulation factor II (thrombin) receptor, mRNA.
3800050−1.503downADCY3109adenylate cyclase 3, mRNA.
5900008−1.504downKLK1111012kallikrein-related peptidase 11, transcript variant 2, mRNA.
5080605−1.504downSNRPA16627small nuclear ribonucleoprotein polypeptide A′, mRNA.
4560541−1.521downMLKL197259mixed lineage kinase domain-like, mRNA.
520682−1.523downCPA451200carboxypeptidase A4, mRNA.
4010296−1.527downRNASE16035ribonuclease, RNase A family, 1 (pancreatic), transcript variant 1, mRNA.
6350161−1.530downLCP13936lymphocyte cytosolic protein 1 (l-plastin), mRNA.
4730605−1.532downAURKA6790aurora kinase A, transcript variant 5, mRNA.
6840075−1.532downNP4860nucleoside phosphorylase, mRNA.
6770187−1.533downSPRR2A6700small proline-rich protein 2A, mRNA.
870131−1.533downHSPA53309heat shock 70 kDa protein 5 (glucose-regulated protein, 78 kDa), mRNA.
1570193−1.535downARHGDIB397Rho GDP dissociation inhibitor (GDI) beta, mRNA.
2450167−1.537downRPL296159ribosomal protein L29, mRNA.
7510709−1.540downCEP5555165centrosomal protein 55 kDa, mRNA.
2350465−1.544downRPL296159ribosomal protein L29, mRNA.
160097−1.546downMELK9833maternal embryonic leucine zipper kinase, mRNA.
3930703−1.547downWDR410785WD repeat domain 4, transcript variant 2, mRNA.
1170066−1.554downSULT2B16820sulfotransferase family, cytosolic, 2B, member 1, transcript variant 1, mRNA.
2070520−1.556downCDCA783879cell division cycle associated 7, transcript variant 1, mRNA.
6550048−1.559downDHCR717177-dehydrocholesterol reductase, mRNA.
5310634−1.566downFASN2194fatty acid synthase, mRNA.
6560494−1.566downARTN9048artemin, transcript variant 2, mRNA.
5860348−1.568downSC4MOL6307sterol-C4-methyl oxidase-like, transcript variant 2, mRNA.
5270112−1.570downHMGCS131573-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (soluble), transcript variant 2, mRNA.
5690274−1.571downMCM64175minichromosome maintenance complex component 6, mRNA.
940487−1.573downFUT32525fucosyltransferase 3 (galactoside 3(4)-l-fucosyltransferase, Lewis blood group), transcript variant 4, mRNA.
5810154−1.580downALOX15B247arachidonate 15-lipoxygenase, type B, transcript variant b, mRNA.
870546−1.581downMAD2L14085MAD2 mitotic arrest deficient-like 1 (yeast), mRNA.
6020139−1.588downKLK75650kallikrein-related peptidase 7, transcript variant 1, mRNA.
4250156−1.589downEBP10682emopamil binding protein (sterol isomerase), mRNA.
10341−1.599downSHMT26472serine hydroxymethyltransferase 2 (mitochondrial), nuclear gene encoding mitochondrial protein, mRNA.
5360678−1.602downDHCR717177-dehydrocholesterol reductase, transcript variant 1, mRNA.
6580059−1.610downUCP27351uncoupling protein 2 (mitochondrial, proton carrier), nuclear gene encoding mitochondrial protein, mRNA.
5090278−1.610downGPX22877glutathione peroxidase 2 (gastrointestinal), mRNA.
3940673−1.617downLOC728285728285similar to keratin associated protein 2-4 (LOC728285), mRNA.
2650564−1.623downRARRES35920retinoic acid receptor responder (tazarotene induced) 3, mRNA.
360367−1.625downPCDH75099protocadherin 7, transcript variant a, mRNA.
7560364−1.635downLOC729779729779misc_RNA (LOC729779), miscRNA.
780528−1.635downCKS21164CDC28 protein kinase regulatory subunit 2, mRNA.
5960224−1.636downPTTG3P26255pituitary tumor-transforming 3 (pseudogene), non-coding RNA.
4730196−1.653downTK17083thymidine kinase 1, soluble, mRNA.
1510296−1.656downASNS440asparagine synthetase, transcript variant 1, mRNA.
1190142−1.657downEMILIN284034elastin microfibril interfacer 2, mRNA.
1170170−1.662downSTC28614stanniocalcin 2, mRNA.
2140128−1.670downSCD6319stearoyl-CoA desaturase (delta-9-desaturase), mRNA.
5360070−1.674downCCNB29133cyclin B2, mRNA.
3990619−1.675downTOP2A7153topoisomerase (DNA) II alpha 170 kDa, mRNA.
3780047−1.679downGBP6163351guanylate binding protein family, member 6, mRNA.
2000148−1.683downIFIT13434interferon-induced protein with tetratricopeptide repeats 1, transcript variant 2, mRNA.
2070494−1.700downPRC19055protein regulator of cytokinesis 1, transcript variant 2, mRNA.
10414−1.704downPTTG19232pituitary tumor-transforming 1, mRNA.
2940110−1.720downUHRF129128ubiquitin-like with PHD and ring finger domains 1, transcript variant 1, mRNA.
1510291−1.733downPTTG19232pituitary tumor-transforming 1, mRNA.
1780446−1.739downPCK25106phosphoenolpyruvate carboxykinase 2 (mitochondrial), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA.
1660521−1.745downSPRR2D6703small proline-rich protein 2D, mRNA.
730689−1.763downLOC652595652595similar to U2 small nuclear ribonucleoprotein A (U2 snRNP-A) (LOC652595), mRNA.
5090754−1.766downKIAA01019768KIAA0101, transcript variant 1, mRNA.
5080139−1.789downPRSS35646protease, serine, 3 (mesotrypsin), mRNA.
3800452−1.805downEMP32014epithelial membrane protein 3, mRNA.
1230047−1.810downCBS875cystathionine-beta-synthase, mRNA.
6370615−1.858downTGM17051transglutaminase 1 (K polypeptide epidermal type I, protein-glutamine-gamma-glutamyltransferase), mRNA.
5310471−1.894downUBE2C11065ubiquitin-conjugating enzyme E2C, transcript variant 6, mRNA.
7380719−1.897downIGFBP63489insulin-like growth factor binding protein 6, mRNA.
940327−1.907downKLK1326085kallikrein-related peptidase 13, mRNA.
520195−1.914downTMEM7984283transmembrane protein 79, mRNA.
4040398−1.954downMAL4118mal, T-cell differentiation protein, transcript variant d, mRNA.
1990630−1.979downTRIB357761tribbles homolog 3 (Drosophila), mRNA.
430446−1.996downKRT813887keratin 81, mRNA.
4260368−2.022downUBE2C11065ubiquitin-conjugating enzyme E2C, transcript variant 3, mRNA.
290767−2.038downKRTDAP388533keratinocyte differentiation-associated protein, mRNA.
6520139−2.046downFGFR32261fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism), transcript variant 2, mRNA.
620102−2.046downMALL7851mal, T-cell differentiation protein-like, mRNA.
5870653−2.050downLOC651397651397misc_RNA (LOC651397), miscRNA.
4050398−2.071downKLK1243849kallikrein-related peptidase 12, transcript variant 1, mRNA.
7330753−2.102downACAT239acetyl-Coenzyme A acetyltransferase 2, mRNA.
4900458−2.147downKRT143861keratin 14 (epidermolysis bullosa simplex, Dowling-Meara, Koebner), mRNA.
540546−2.283downKRT43851keratin 4, mRNA.
1500010−2.322downCDC20991cell division cycle 20 homolog (S. cerevisiae), mRNA.
6550356−2.430downSPRR2C6702small proline-rich protein 2C (pseudogene), non-coding RNA.
4850674−2.452downPSAT129968phosphoserine aminotransferase 1, transcript variant 2, mRNA.
5890400−2.577downSPRR2E6704small proline-rich protein 2E, mRNA.
240086−2.608downPHGDH26227phosphoglycerate dehydrogenase, mRNA.
7650441−2.696downFGFBP19982fibroblast growth factor binding protein 1, mRNA.
5810546−2.894downSPRR2E6704small proline-rich protein 2E, mRNA.
7330184−2.933downSPRR1A6698small proline-rich protein 1A, mRNA.
2230035−2.936downKRT133860keratin 13, transcript variant 2, mRNA.
4610131−3.284downSPRR36707small proline-rich protein 3, transcript variant 1, mRNA.
In red up-regulated and in green down-regulated genes in BE63/3 cells treated with 100 nM EVE compared to CTR.
Table
Table 2. List of pathways differentially regulated after 100 nM EVE.
Table 2. List of pathways differentially regulated after 100 nM EVE.
PathwaysAdj. p ValueAssociated Genes
Epidermis development1.24 × 10−6ALOX15B, CTGF, FOXQ1, FZD6, KLK7, KRT14, RNASE1, SPRR1A, SPRR2A, SPRR2D, SPRR2E, SPRR3, TGM1, TMEM79, TXNIP
Keratinization5.22 × 10−6SPRR1A, SPRR2A, SPRR2D, SPRR2E, SPRR3, TGM1, TMEM79
Negative regulation of cell division2.58 × 10−5CDC20, FGFR3, MAD2L1, PTTG1, PTTG3P, RGCC, TXNIP, UBE2C
Negative regulation of mitotic nuclear division2.81 × 10−5CDC20, FGFR3, MAD2L1, PTTG1, PTTG3P, RGCC, UBE2C
Keratinocyte differentiation3.05 × 10−5ALOX15B, SPRR1A, SPRR2A, SPRR2D, SPRR2E, SPRR3, TGM1, TMEM79, TXNIP
L-serine metabolic process3.54 × 10−5CBS, PHGDH, PSAT1, SHMT2
Epidermal cell differentiation9.21 × 10−5ALOX15B, RNASE1, SPRR1A, SPRR2A, SPRR2D, SPRR2E, SPRR3, TGM1, TMEM79, TXNIP
L-serine biosynthetic process9.75 × 10−5PHGDH, PSAT1, SHMT2
Negative regulation of nuclear division1.10 × 10−4CDC20, FGFR3, MAD2L1, PTTG1, PTTG3P, RGCC, UBE2C
Skin development1.82 × 10−4ALOX15B, FOXQ1, FZD6, SPRR1A, SPRR2A, SPRR2D, SPRR2E, SPRR3, TGM1, TMEM79, TXNIP
Peptide cross-linking2.05 × 10−4SPRR1A, SPRR2A, SPRR2D, SPRR2E, SPRR3, TGM1
Serine family amino acid biosynthetic process3.55 × 10−4CBS, PHGDH, PSAT1, SHMT2
Regulation of collagen metabolic process5.84 × 10−4CTGF, F2R, FAP, IL6, RGCC
Regulation of multicellular organismal metabolic process6.51 × 10−4CTGF, F2R, FAP, IL6, RGCC
Steroid biosynthesis6.77 × 10−4CYP24A1, DHCR7, EBP, MSMO1
Chromosome separation0.00192CDC20, MAD2L1, PTTG1, PTTG3P, TOP2A, UBE2C
Negative regulation of mitotic sister chromatid separation0.00199CDC20, MAD2L1, PTTG1, PTTG3P, UBE2C
Collagen metabolic process0.00200COL12A1, CTGF, F2R, FAP, IL6, MMP12, RGCC
Negative regulation of mitotic sister chromatid segregation0.00231CDC20, MAD2L1, PTTG1, PTTG3P, UBE2C
Multicellular organismal macromolecule metabolic process0.00248COL12A1, CTGF, F2R, FAP, IL6, MMP12, RGCC
Negative regulation of sister chromatid segregation0.00267CDC20, MAD2L1, PTTG1, PTTG3P, UBE2C
Negative regulation of chromosome segregation0.00267CDC20, MAD2L1, PTTG1, PTTG3P, UBE2C
Regulation of nuclear division0.00302AURKA, CDC20, FGFR3, MAD2L1, PTTG1, PTTG3P, RGCC, UBE2C
Multicellular organismal metabolic process0.00456COL12A1, CTGF, F2R, FAP, IL6, MMP12, RGCC
Regulation of collagen biosynthetic process0.00457CTGF, F2R, IL6, RGCC
Mitotic sister chromatid separation0.00664CDC20, MAD2L1, PTTG1, PTTG3P, UBE2C
Regulation of mitotic sister chromatid segregation0.00834CDC20, MAD2L1, PTTG1, PTTG3P, UBE2C
Sister chromatid segregation0.00851CDC20, CEP55, MAD2L1, PTTG1, PTTG3P, TOP2A, UBE2C
Glycine, serine and threonine metabolism0.00873CBS, PHGDH, PSAT1, SHMT2
Collagen biosynthetic process0.00873CTGF, F2R, IL6, RGCC
Oocyte meiosis0.01153ADCY3, AURKA, CCNB2, CDC20, MAD2L1, PTTG1
Regulation of sister chromatid segregation0.01277CDC20, MAD2L1, PTTG1, PTTG3P, UBE2C
Negative regulation of chromosome organization0.01396ARTN, CDC20, MAD2L1, PTTG1, PTTG3P, UBE2C
PERK-mediated unfolded protein response0.01404ASNS, ATF3, HSPA5
Regulation of stress fiber assembly0.01630CTGF, RGCC, RNASE1
FoxO signaling pathway0.01634CCNB2, GABARAPL1, IL6, IRS2, PCK2, SGK1
Anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolic process0.01664AURKA, CDC20, MAD2L1, PTTG1, UBE2C
Alpha-amino acid biosynthetic process0.01664ASNS, CBS, PHGDH, PSAT1, SHMT2
Positive regulation of collagen biosynthetic process0.02234CTGF, F2R, RGCC
Regulation of systemic arterial blood pressure by circulatory renin-angiotensin0.02412CPA4, F2R, MMP12
Positive regulation of multicellular organismal metabolic process0.02412CTGF, F2R, RGCC
Secondary alcohol biosynthetic process0.02578DHCR7, EBP, HMGCS1, MSMO1
Regulation of chromosome segregation0.02590CDC20, MAD2L1, PTTG1, PTTG3P, UBE2C
Negative regulation of proteasomal ubiquitin-dependent protein catabolic process0.03145CDC20, MAD2L1, UBE2C
In red up-regulated and in green down-regulated genes in BE63/3 cells treated with 100 nM EVE compared to CTR.
Table
Table 3. List of probe sets differentially expressed after treatment with 5 nM EVE.
Table 3. List of probe sets differentially expressed after treatment with 5 nM EVE.
Probe IDFold ChangeRegulationSymbolEntrez Gene IDDefinition
22300357.508upKRT133860keratin 13, transcript variant 2, mRNA.
65107543.841upALDH1A1216aldehyde dehydrogenase 1 family, member A1, mRNA.
10704773.395upALDH1A1216aldehyde dehydrogenase 1 family, member A1, mRNA.
5405462.749upKRT43851keratin 4, mRNA.
19901422.644upC20orf11492747chromosome 20 open reading frame 114, mRNA.
59003682.385upMSMB4477microseminoprotein, beta-, transcript variant PSP94, mRNA.
46101312.358upSPRR36707small proline-rich protein 3, transcript variant 1, mRNA.
31901102.194upMSMB4477microseminoprotein, beta-, transcript variant PSP94, mRNA.
6303152.151upDHRS910170dehydrogenase/reductase (SDR family) member 9, transcript variant 1, mRNA.
54205772.149upCLCA422802chloride channel, calcium activated, family member 4, mRNA.
55603692.107upALDH3A1218aldehyde dehydrogenase 3 family, memberA1, mRNA.
41505981.990upMSMB4477microseminoprotein, beta-, transcript variant PSP57, mRNA.
18204141.897upATP12A479ATPase, H+/K+ transporting, nongastric, alpha polypeptide, mRNA.
35207091.888upADH7131alcohol dehydrogenase 7 (class IV), mu or sigma polypeptide, mRNA.
71604681.807upDHRS910170dehydrogenase/reductase (SDR family) member 9, transcript variant 1, mRNA.
53106461.795upAKR1B1057016aldo-keto reductase family 1, member B10 (aldose reductase), mRNA.
42500921.749upC10orf99387695chromosome 10 open reading frame 99, mRNA.
1103721.748upCSTA1475cystatin A (stefin A), mRNA.
37106711.712upKRT153866keratin 15, mRNA.
17706031.705upTCN16947transcobalamin I (vitamin B12 binding protein, R binder family), mRNA.
61005371.655upFAM3D131177family with sequence similarity 3, member D, mRNA.
45404001.623upCYP4B11580cytochrome P450, family 4, subfamily B, polypeptide 1, transcript variant 2, mRNA.
29000501.611upGSTA12938glutathione S-transferase alpha 1, mRNA.
15101701.565upNLRP255655NLR family, pyrin domain containing 2, mRNA.
58204001.526upCYP4B11580cytochrome P450, family 4, subfamily B, polypeptide 1, mRNA.
1305611.525upGSTA42941glutathione S-transferase A4, mRNA.
38502461.513upHOPX84525HOP homeobox, transcript variant 3, mRNA.
7200612−1.522downLOC730417730417hypothetical protein LOC730417, mRNA.
1510296−1.556downASNS440asparagine synthetase, transcript variant 1, mRNA.
3290390−1.563downLOC729841729841misc_RNA, miscRNA.
7380193−1.574downARPC310094actin related protein 2/3 complex, subunit 3, 21 kDa, mRNA.
130717−1.610downARPC1B10095actin related protein 2/3 complex, subunit 1B, 41 kDa, mRNA.
430446−1.689downKRT813887keratin 81, mRNA.
In red up-regulated and in green down-regulated genes in BE63/3 cells treated with 5 nM EVE compared to CTR.
Table
Table 4. List of pathways differentially regulated after treatment with 5 nM EVE.
Table 4. List of pathways differentially regulated after treatment with 5 nM EVE.
PATHWAYSAdj. p ValueAssociated Genes Found
Retinol metabolism8.58 × 10−5ADH7, ALDH1A1, DHRS9
Metabolism of xenobiotics by cytochrome P4501.48 × 10−5ADH7, ALDH3A1, GSTA1, GSTA4
Drug metabolism1.37 × 10−5ADH7, ALDH3A1, GSTA1, GSTA4
Retinoid metabolic process1.41 × 10−5ADH7, AKR1B10, ALDH1A1, DHRS9
Chemical carcinogenesis1.96 × 10−5ADH7, ALDH3A1, GSTA1, GSTA4
Cellular aldehyde metabolic process2.60 × 10−5ADH7, AKR1B10, ALDH1A1, ALDH3A1
Primary alcohol metabolic process3.30 × 10−6ADH7, AKR1B10, ALDH1A1, DHRS9
Retinol metabolic process1.99 × 10−5ADH7, ALDH1A1, DHRS9
In red up-regulated genes in BE63/3 cells treated with 5 nM EVE compared to CTR.
Table
Table 5. List of microRNAs differentially regulated after treatment with 100 nM EVE.
Table 5. List of microRNAs differentially regulated after treatment with 100 nM EVE.
Systematic NameRegulationFold Change
hsa-miR-8485up5.372
hsa-miR-937-5pup1.787
hsa-miR-5194up1.694
Table
Table 6. List of microRNAs differentially regulated after treatment with 5 nM EVE.
Table 6. List of microRNAs differentially regulated after treatment with 5 nM EVE.
Systematic NameRegulationFold Change
hsa-miR-8485up9.183
hsa-miR-4730up2.900
hsa-miR-5194up2.732
hsa-miR-6716-3pup2.561
Table
Table 7. miRNA/mRNA pairs matched on the basis of mRNA and miRNA profiling results.
Table 7. miRNA/mRNA pairs matched on the basis of mRNA and miRNA profiling results.
Cell TreatmentsmiRNAFold ChangemRNA TargetGene Name
EVE 5 nMmiR-84859.183CYP4B1cytochrome P450, family 4, subfamily B, polypeptide 1
miR-51942.732ARPC3actin related protein 2/3 complex, subunit 3, 21 kDa
EVE 100 nMmiR-84855.372CYP24A1cytochrome P450, family 24, subfamily A, polypeptide 1
KAL1Kallmann syndrome 1 sequence
UBL3ubiquitin-like 3
IRS2insulin receptor substrate 2
CTGFconnective tissue growth factor
LBHlimb bud and heart development
FLRT2fibronectin leucine rich transmembrane protein 2
CDH6cadherin 6, type 2, K-cadherin (fetal kidney)
CYBRD1cytochrome b reductase 1
LETMD1LETM1 domain containing 1
FGFR3fibroblast growth factor receptor 3
CPA4carboxypeptidase A4
AURKAaurora kinase A
CBScystathionine-beta-synthase
MAD2L1MAD2 mitotic arrest deficient-like 1 (yeast)
ADCY3adenylate cyclase 3
TMEM79transmembrane protein 79
IFIT1interferon-induced protein with tetratricopeptide repeats 1
PTTG1pituitary tumor-transforming 1
PCDH7protocadherin 7
miR-937-5p1.787CDH6cadherin 6, type 2, K-cadherin (fetal kidney)
KIAA0101KIAA0101
EMILIN2elastin microfibril interfacer 2
miR-51941.694KLHL24kelch-like family member 24
FAPfibroblast activation protein, alpha
LBHlimb bud and heart development
PIM1pim-1 oncogene
FLRT2fibronectin leucine rich transmembrane protein 2
LETMD1LETM1 domain containing 1
FGFR3fibroblast growth factor receptor 3
KIAA0101KIAA0101
RARRES3retinoic acid receptor responder (tazarotene induced) 3
ARTNartemin
IGFBP6insulin-like growth factor binding protein 6
LCP1lymphocyte cytosolic protein 1 (L-plastin)
MALLsmall integral membrane protein 5
SCDLSM14B, SCD6 homolog B (S. cerevisiae)
IFIT1interferon-induced protein with tetratricopeptide repeats 1
In red up-regulated and in green down-regulated genes in BE63/3 cells treated with EVE (5 or 100 nM) compared to CTR.

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Granata, S.; Santoro, G.; Masola, V.; Tomei, P.; Sallustio, F.; Pontrelli, P.; Accetturo, M.; Antonucci, N.; Carratù, P.; Lupo, A.; et al. In Vitro Identification of New Transcriptomic and miRNomic Profiles Associated with Pulmonary Fibrosis Induced by High Doses Everolimus: Looking for New Pathogenetic Markers and Therapeutic Targets. Int. J. Mol. Sci. 2018, 19, 1250. https://doi.org/10.3390/ijms19041250

AMA Style

Granata S, Santoro G, Masola V, Tomei P, Sallustio F, Pontrelli P, Accetturo M, Antonucci N, Carratù P, Lupo A, et al. In Vitro Identification of New Transcriptomic and miRNomic Profiles Associated with Pulmonary Fibrosis Induced by High Doses Everolimus: Looking for New Pathogenetic Markers and Therapeutic Targets. International Journal of Molecular Sciences. 2018; 19(4):1250. https://doi.org/10.3390/ijms19041250

Chicago/Turabian Style

Granata, Simona, Gloria Santoro, Valentina Masola, Paola Tomei, Fabio Sallustio, Paola Pontrelli, Matteo Accetturo, Nadia Antonucci, Pierluigi Carratù, Antonio Lupo, and et al. 2018. "In Vitro Identification of New Transcriptomic and miRNomic Profiles Associated with Pulmonary Fibrosis Induced by High Doses Everolimus: Looking for New Pathogenetic Markers and Therapeutic Targets" International Journal of Molecular Sciences 19, no. 4: 1250. https://doi.org/10.3390/ijms19041250

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