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

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.

Keywords: epithelial to mesenchymal transition; everolimus; mTOR inhibitor; miRNome; pulmonary fibrosis; transcriptomics.

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 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 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 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 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.