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. 2016 Jun 10;13(9):837–847. doi: 10.1080/15476286.2016.1197484

RPTOR, a novel target of miR-155, elicits a fibrotic phenotype of cystic fibrosis lung epithelium by upregulating CTGF

Motohiro Tsuchiya a,b, Swathi Kalurupalle b, Parameet Kumar b, Sarani Ghoshal b, Yongqing Zhang c, Elin Lehrmann c, Kevin G Becker c, Myriam Gorospe c, Roopa Biswas b
PMCID: PMC5013987  PMID: 27284727

ABSTRACT

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the most frequent of which is F508del-CFTR. CF is characterized by excessive secretion of pro-inflammatory mediators into the airway lumen, inducing a highly inflammatory cellular phenotype. This process triggers fibrosis, causing airway destruction and leading to high morbidity and mortality. We previously reported that miR-155 is upregulated in CF lung epithelial cells, but the molecular mechanisms by which miR-155 affects the disease phenotype is not understood. Here we report that RPTOR (regulatory associated protein of mTOR, complex 1) is a novel target of miR-155 in CF lung epithelial cells. The suppression of RPTOR expression and subsequent activation of TGF-β signaling resulted in the induction of fibrosis by elevating connective tissue growth factor (CTGF) abundance in CF lung epithelial cells. Thus, we propose that miR-155 might regulate fibrosis of CF lungs through the increased CTGF expression, highlighting its potential value in CF therapy.

KEYWORDS: CTGF, Cystic fibrosis, inflammation, lung epithelium, microRNA, miR-155, RPTOR, TGF-β signaling

Introduction

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of a chloride ion channel called cystic fibrosis transmembrane conductance regulator (CFTR).1 CFTR mutations, of which the most frequent is F508del-CFTR, cause a massive pro-inflammatory phenotype in the lung, which manifests in the airway by high levels of IL-8 and other pro-inflammatory cytokines and chemokines.2-4 CF lung disease is the major life-threatening factor for CF patients. CF lung has been shown to be inherently inflammatory, and CF patients eventually develop pulmonary fibrosis and airway remodeling and bronchiectasis.5 We have previously reported that the microRNA (miR)-155 is elevated in CF lungs and is a major inducer of the inflammatory phenotype of CF. However, the mechanism by which miR-155 affects CF-specific target genes and CF disease is not understood.

MicroRNAs (miRNAs, miRs), a class of ∼22-nucleotide-long endogenous RNA molecules, have been shown to negatively regulate gene expression by promoting mRNA degradation and/or translational suppression.6-9 miRNAs are key molecules in disease development and are therefore important therapeutic targets. Several studies have analyzed the role of miRNAs in CF,10-16 however little is known about regulation of inflammation and fibrosis by miRNAs. Lower expression of miR-126 in CF was shown to affect the inflammatory phenotype of CF via upregulation of TOM1.11 Previously we showed that miR-155 is upregulated in CF lung epithelial cells, both cell lines and primary cells isolated from lung explants of CF patients. We demonstrated that the elevated expression of miR-155 induced increased IL-8 expression by suppressing SHIP1 production, thereby activating the PI3K/Akt signaling pathway.12

Here we report for the first time that miR-155 regulates expression of connective tissue growth factor (CTGF), which is known to stimulate tissue fibrosis.17,18 We have identified a novel target gene of miR-155 in CF lung epithelial cells, regulatory associated protein of mTOR, complex 1 (RPTOR). RPTOR is involved in the regulation of the mammalian target of rapamycin complex 1 (mTORC1) activity which regulates cell growth and survival. We find that elevated miR-155 expression resulted in decreased RPTOR expression in CF lung epithelial cells which in turn activated TGF-β signaling and increased expression of CTGF. Anti-CTGF therapy is currently used in clinical trials of diseases including diabetic kidney disease 19 as well as in disease models of pulmonary fibrosis.20 This is the first report to show the regulation of CF disease through miR-155-RAPTOR-CTGF axis. Our data suggests CTGF as a potential target for CF therapy.

Results

Identification of candidate targets of miR-155 in CF

We previously demonstrated that elevated expression of miR-155 in CF bronchial epithelial cells induces expression of the pro-inflammatory cytokine IL-8 via increased PI3K/Akt signaling.12 Therefore, we sought disease-specific targets of miR-155 in CF. To identify miR-155 targets, we conducted concurrent analyses of mRNA expression profile (ILLUMINA bead arrays) under 2 different conditions. For the first strategy, we employed a biochemical method developed by Lal et al.21 A 3′-end-biotinylated miR-155 (10 nM) or biotinylated cel-miR-67 control was incubated with CFTR-repaired IB3-1/S9 lung epithelial cells for 24 hours. Subsequently, the cell lysates were enriched for mRNA:miR-155 complexes using streptavidin bead-based affinity purification and analyzed the bound mRNAs. For the second strategy, we transfected IB3-1/S9 lung epithelial cells with miR-155-mimic (10 nM) or control mimic and analyzed the mRNA profile 48 hours later.

Potential targets of miR-155 are expected to be enriched in the affinity-based isolation employing biotinylated-miR-155 and should be down-regulated in cells in miR-155 is overexpressed. Thus, we sought mRNAs which had positive Z-scores from the former group and negative Z-scores from the latter group. Fig. 1A depicts the overlap in the number of mRNAs identified by these approaches as well as in silico analyses of the predicted targets of miR-155 (TargetScan 6.2). Among the 152 mRNAs which satisfied all 3 criteria, we selected Casein Kinase 1 Gamma 2 (CSNK1G2), Interleukin enhancer-binding factor 3 (ILF3/NF90) and Regulatory-associated Protein of mTOR (RPTOR) mRNAs, based on at least one false discovery rate (FDR) less than 0.3, and any involvement in signaling pathways or other related mechanisms in inflammatory processes (Fig. 1B).22-24 Analyses of these candidate targets in CF cells indicated that CSNK1G2 and IF3/NF90 mRNA levels were comparable in CF and control cells. However, RPTOR mRNA was significantly lower in IB3-1CF cells than in IB3-1/S9 control cells and thus subsequent analyses focused on the role RPTOR in regulating the CF disease phenotype.

Figure 1.

Figure 1.

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Analyses of candidate target genes of miR-155 targets. (A) Venn diagram representing the number of genes enriched by affinity-based isolation, those downregulated in control cells overexpressing miR-155 mimic, and those predicted as targets of miR-155. (B) Fold enrichment or suppression of top 3 candidate mRNA targets of miR-155 (Z-scores). The respective qPCR data for the 3 mRNAs in IB3-1 cells compared to IB3-1/S9 cells are also indicated. The data are representative of 2 or more independent experiments (Values indicate 2-AvgΔΔCt±StdevΔΔCt. *p < 0.05).

Validation of RPTOR as a direct target of miR-155

To validate RPTOR as a direct target of miR-155, we first analyzed the expression of RPTOR protein in IB3-1 and IB3-1/S9 cells. As shown in the Fig. 2A, RPTOR protein expression was significantly lower in IB3-1 CF cells compared to IB3-1/S9 CFTR-repaired control cells. Subsequently, we analyzed effect of modulating miR-155 expression on RPTOR protein level. We found that IB3-1 CF cells in which miR-155 was suppressed with anti-miR-155 exhibited increased RPTOR protein expression compared to controls (Fig. 2B). Consistently, overexpression of miR-155 in IB3-1/S9 control cells by introducing a miR-155 mimic reduced RPTOR protein levels (Fig. 2C).

Figure 2.

Figure 2.

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Effect of miR-155 on RPTOR expression in CF. (A) RPTOR protein expression in IB3-1/S9 (control) and IB3-1 (CF) cells was analyzed by Western blot. (B) IB3-1 cells (2.5 × 105) were treated with anti-miR-155 (150 nM, 48 hours) or negative control (anti-miR-CTRL) and RPTOR protein was analyzed by Western blot. (C) RPTOR expression levels were also analyzed (Western blot) in IB3-1/S9 cells (2.5 × 105) overexpressing pre-miR-155 (50 nM, 48 hours) or negative control (pre-miR-CTRL). The graph below each of the immunoblots represents respective relative protein quantification (using ImageJ). The data are the means ± SEM from 3 independent experiments.

To further test if RPTOR was a direct miR-155 target, we cloned the region of the RPTOR 3′-UTR downstream of a luciferase reporter gene in the pMIR-Report vector (Fig. 3A); if the 3′-UTR sequence is a valid miRNA target, luciferase activity would decline. A control pMIR-Report β-gal vector was transfected simultaneously to normalize the transfection efficiency. pMIR-Report derivatives in which the RPTOR mRNA 3′-UTR target sequences were mutated was included as controls for these assays. The vectors (WT or mutant) were transfected into IB3-1 CF cells incubated with anti-miR-155 (Fig. 3B) or into IB3-1/S9 cells treated with miR-155 mimic (Fig. 3C) and respective scrambled controls. Luciferase activity was significantly elevated (∼37.2%) by suppressing miR-155 in IB3-1 CF cells and reduced (∼32.7%) by overexpressing miR-155 in IB3-1/S9 control cells when the WT RPTOR target site for miR-155 is used. In contrast, the reporter harboring the mutant miR-155 target sequence showed unaltered luciferase activity. Thus RPTOR is validated as a novel direct target of miR-155 in CF lung epithelial cells.

Figure 3.

Figure 3.

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Validation of RPTOR mRNA as a direct target of miR-155. (A) The miR-155 target RPTOR 3′-UTR sequences (WT) as well as that of the mutated-derivative (Mut, underlined sequence) are shown. Bold letters indicate miR-155 binding site predicted by TargetScan. (B) Luciferase reporter assays were performed in IB3-1 CF cells transfected with pMIR-Report vectors, either controls or those containing RPTOR 3′-UTR target sequences of miR-155 (both WT and mutant), and anti-miR-155 or anti-miR-CTRL (150 nM, 24 h). (C) Similar assays were performed in IB3-1/S9 CFTR-repaired control cells in the presence or absence of pre-miR-155 or pre-miR-CTRL (50 nM, 24 h). The data reflect averages of at least 3 independent experiments (*P < 0.05, **P > 0.05). Luc-only: pMIR-Report vector plasmid without RPTOR sequence.

Activation of TGF-β signaling pathway after RPTOR inhibition

We next investigated the effect of RPTOR protein on the CF disease phenotype. To examine the role of RPTOR, we used the pharmacological inhibitor rapamycin, which is known to bind to FKBP12 and thus prevent RPTOR from binding to mTOR to form mTORC1.25 Treatment of IB3-1/S9 CFTR-repaired control cells with rapamycin (50 nM, 48 h) results in reduced phosphorylation of mTOR protein (Fig. 4A). As expected, the IB3-1 CF cells exhibit decreased mTOR phosphorylation compared to IB3-1/S9 control cells (Fig. 4B). Since, rapamycin has been shown to induce TGF-β, a well-studied profibrogenic cytokine,26,27 we measured TGFB1 mRNA levels in CF lung epithelial cells and also in control cells treated with rapamycin. As depicted in Fig. 4C, rapamycin treatment significantly induced TGFB1 mRNA (encoding TGF-β) in IB3-1/S9 cells. Consistently, TGFB1 mRNA level were relatively higher in IB3-1 CF cells compared to IB3-1/S9 control cells (Fig. 4D).

Figure 4.

Figure 4.

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Effect of rapamycin on downstream signaling pathways in CF cells. (A) IB3-1/S9 cells (2.5 × 105) were treated with rapamycin (50 nM, 48 h) or equal volume of DMSO (0.005 vol%) and expression of phosphorylated mTOR protein was assessed by protein gel blot analysis. (B) Simultaneously, phosphorylated mTOR protein was similarly analyzed in IB3-1 cells and IB3-1/S9 cells. The graph below respective immunoblot represents relative protein quantification normalized to GAPDH protein levels. The data is average of 3 independent experiments (mean ± SEM). TGFβ mRNA levels were analyzed by qPCR (C) in IB3-1/S9 cells (2.5 × 105) treated with rapamycin (50 nM, 48 h) or with equal volume of DMSO (0.005 vol%) and (D) in IB3-1 CF cells compared to IB3-1/S9 control cells. The data is averages of at least 3 independent experiments (values indicate 2-AvgΔΔCt±StdevΔΔCt, *P < 0.05).

Next we examined whether TGF-β signaling was activated by rapamycin in CF lung epithelial cells. As shown in Fig. 5A, IB3-1 CF cells exhibited increased phosphorylation of SMAD2 compared to IB3-1/S9 control cells. Moreover activation of TFG-β signaling in IB3-1/S9 control cells by treatment with rapamycin resulted in increased phosphorylation of SMAD2 (Fig. 5B). Since RPTOR expression is suppressed by miR-155, we examined the effects of miR-155 levels on SMAD2 phosphorylation. As depicted in Fig. 6A, inhibition of miR-155 in IB3-1 CF cells resulted in decreased SMAD2 phosphorylation. Conversely, overexpression of miR-155 in IB3-1/S9 control cells induced SMAD2 phosphorylation (Fig. 6B). Thus, our data indicate that elevated expression of miR-155 in IB3-1 CF lung epithelial cells resulted in reduced RPTOR protein levels, and subsequently increased SMAD2 phosphorylation.

Figure 5.

Figure 5.

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Effect of RPTOR inhibition on TGFβ signaling pathway in CF cells. Expression of phosphorylated SMAD2 protein was analyzed by western blot (A) in IB3-1/S9 and IB3-1 cells and (B) in IB3-1/S9 cell treated with rapamycin (50 nM, 48 h). The graph below the immunoblot represents relative protein quantification normalized to GAPDH protein levels. The data are the means ± SEM from 3 independent experiments.

Figure 6.

Figure 6.

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Effect of miR-155 on TGFβ signaling pathway in CF cells. Expression of phosphorylated SMAD2 protein was analyzed by protein gel blot (A) in IB3-1 cells (2.5 × 105) treated with negative control (anti-miR-CTRL) or anti-miR-155 (150 nM 48 h) and (B) in IB3-1/S9 cells (2.5 × 105) treated pre-miR-155 (50 nM, 48 hours) or negative control (pre-miR-CTRL). The graph below respective immunoblot represents relative protein quantification normalized to GAPDH expression levels. The data is representative of 3 independent experiments (mean ± SD).

CTGF is induced in CF lung epithelial cells

In light of the finding that miR-155 induces TGF-β signaling pathway in CF lung epithelial cells, we analyzed downstream targets which might promote the fibrotic disease phenotype of CF. We thus examined the expression of connective tissue growth factor (CTGF, also known as CCN2), as it is known to be induced by rapamycin through TGF-β signaling pathway.28 CTGF is induced by physical, chemical or biological external stimuli (such as mechanical stretch, hypoxia or inflammation), and activates fibroblasts for more collagen synthesis, which leads to fibrosis.17,18 Even though CTGF expression is high in several respiratory diseases such as asthma and emphysema,29,30 its role in CF has not been examined. Interestingly, we found that IB3-1 CF cells exhibited increased expression of CTGF compared to IB3-1/S9 control cells (Figs. 7A and 7B). Moreover, treatment of IB3-1/S9 control cells with rapamycin also induced CTGF mRNA and CTGF protein expressions (Fig. 7C and 7D).

Figure 7.

Figure 7.

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Analysis of CTGF expression in CF lung epithelial cells. CTGF expression was analyzed in IB3-1/S9 control cells and IB3-1 CF cells: (A) mRNA by RT-qPCR analysis and (B) protein by western blot analysis. Subsequently, CTGF expression was also analyzed in IB3-1/S9 cells (2.5 × 105) treated with rapamycin (50 nM, 48 h) or the equal volume of DMSO (0.005 vol %): (C) mRNA by qPCR analysis and (D) protein by protein gel blot analysis. The graph below respective immunoblot represents relative protein quantification normalized to GAPDH expression levels. The data are the means ± SEM from 3 independent experiments.

Next, we analyzed the role of TGF-β signaling pathway in the regulation of CTGF expression. Following treatment of IB3-1 CF cells with TGF-β signaling inhibitor SB431542 for 48 hours resulted in reduced phosphorylation of SMAD2 (Fig. 8A) and significant suppression of CTGF expression (Fig. 8B).

Figure 8.

Figure 8.

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Effect of TGF-β signaling pathway on CTGF expression in CF lung epithelial cells. (A) Expression of phosphorylated SMAD2 protein was analyzed by western blot in IB3-1 cells (2.5 × 105) treated with SB431542 (2 μM, 48 h) or the equal volume of DMSO (0.02 vol %). (B) CTGF expression level was analyzed by protein gel blot in IB3-1 cells (2.5 × 105) treated with SB431542 (2 μM, 48 h) or the equal volume of DMSO (0.02 vol %). The graph below the immunoblot represents relative protein quantification normalized to GAPDH expression levels. The data are the means ± SEM from 3 independent experiments.

To establish if CTGF expression in CF lung epithelial cells is affected by miR-155, we modulated miR-155 levels in CF and control cells. We find that suppressing miR-155 in IB3-1 CF cells reduced CTGF expression (Fig. 9A) and conversely, overexpressing miR-155 in IB3-1/S9 control cells induced CTGF (Fig. 9B).

Figure 9.

Figure 9.

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Effect of miR-155 on CTGF expression in CF lung epithelial cells. Expression of CTGF protein was analyzed by western blot (A) in IB3-1 cells (2.5 × 105) treated with negative control (anti-miR-CTRL) or anti-miR-155 (150 nM 48 h) and (B) in IB3-1/S9 cells (2.5 × 105) treated pre-miR-155 (50 nM, 48 hours) or negative control (pre-miR-CTRL). The graph below respective immunoblot represents relative protein quantification normalized to GAPDH expression levels. The data are the means ± SEM from 3 independent experiments. (C) Using Illumina bead arrays, RPTOR and CTGF mRNA levels were analyzed in primary CF HBE cells (n = 4) compared to control NHBE cells (n = 2) (*p < 0.05).

We further analyzed the expression of RPTOR and CTGF mRNA in vivo in primary CF lung epithelial cells (CF HBE, n = 4). As shown in Fig. 9C, RPTOR mRNA was significantly reduced in CF HBE cells compared to normal human bronchial epithelial cells (NHBE, n = 2). Consistently, we observed significantly higher levels of CTGF mRNA in CF HBE cells compared to NHBE cells. Based on our data, we thus propose that miR-155 regulates the fibrotic phenotype of CF through the RPTOR-TGF-β-CTGF axis (Fig. 10).

Figure 10.

Figure 10.

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Schematic mechanism of CTGF induction in CF lung epithelial cells. Increased expression of miR-155 in CF cells suppresses RPTOR, a direct target of miR-155. Suppression of RPTOR, by elevated miR-155 expression in CF cells or pharmacologically by rapamycin stimulates TGF-β signaling pathway through SMAD2 phosphorylation, and promotes upregulation of CTGF. Consistently, pharmacological inhibition of TGF-β signaling (by SB431542) also results in suppression of CTGF.

Discussion

CF is characterized by a massive pro-inflammatory phenotype in the lung, associated with high levels of IL-8 and other pro-inflammatory cytokines and chemokines in the airways. We previously reported that increased expression of miR-155 in CF drives the pro-inflammatory disease phenotype in CF by hyper-inducing IL-8 levels.12 Here we identified RPTOR as a novel inflammatory target of miR-155 in CF lung epithelial cells. We demonstrate for the first time that reduced expression of RPTOR in CF increases the expression CTGF through activation of the TGF-β signaling pathway.

We used an in vivo affinity-based isolation technique to identify candidate targets of miR-155 in CF lung epithelial cells31 and performed conventional mRNA expression profiling in control cells overexpressing miR-155. Based on these data and the predicted targets of miR-155 (TargetScan),32 we identified RPTOR mRNA as a potential direct target of miR-155. We found evidence that RPTOR might regulate the disease phenotype of CF by modulating CTGF expression levels. Our findings are consistent with earlier reports indicating that miR-155 affects mTORC1 and mTORC2 pathways.33 Although the primary focus of this earlier study was on the expression levels of Rheb, mTOR and RICTOR, RPTOR protein abundance was also decreased in response to miR-155 overexpression.33 The mTORC1 pathway has been shown to be upregulated in airway neutrophils compared with peripheral neutrophils in CF patients,34 but the role of RPTOR in CF lung disease had not been analyzed. Here we report for the first time that RPTOR expression is reduced in CF cells.

Since inhibition of RPTOR induces production of TGF-β, a well-studied profibrogenic cytokine, we analyzed the effect of modulating TGF-β activity on downstream targets. We demonstrated the elevated expression of CTGF in CF. CTGF, a fibrotic factor, has been reported to be upregulated in other inflammatory diseases such as COPD and asthma, promoting increased fibrogenesis and remodeling of the airway tract.29,30 Earlier studies have shown that rapamycin increased CTGF expression via TGF-β signaling pathway in lung epithelial cells and lung fibroblasts.28,35 These studies also support our finding that CTGF may play a role in CF lung fibrosis through elevated miR-155 expression and consequent suppression of RPTOR levels.

TGF-β is also known to stimulate other signaling pathways. It has been shown that rapamycin treatment increased CTGF through TGF-β, but independent of SMAD phosphorylation through the PI3K/Akt pathway.35 Our study only examined how phosphorylation of SMAD2 is induced through activation of TGF-β, resulting in increased expression of CTGF in CF lung epithelial cells. Our previous report demonstrated that PI3K/Akt pathway is activated in CF through elevated expression of miR-155; therefore, it is also possible that these mechanisms together are contributing to the fibrotic phenotype of CF lung disease. Our results suggests novel therapeutic targets for CF. High CTGF expression has been reported in several diseases other than CF, and are shown to be related to disease progression.17 Thus ongoing clinical trials against CTGF in these diseases 17 could be extended to CF therapy.

Materials and methods

Biotinylated microRNA-155 transfection

IB3-1 CF lung epithelial cells and the control CFTR-repaired IB3-1/S9 cells were maintained in LHC-8 serum-free medium (Invitrogen, 12678-017) in humidified 5% CO2. 3.0 × 106 IB3-1/S9 cells were plated on one 100-mm dish and incubated in LHC-8 media for 24 hours. Twenty-four hours later, 10 nM of 3′-biotinylated miR-155 (Dharmacon) or control 5′-biotinylated cel-miR-67 (a gift from Ashish Lal, NCI/NIH) were transfected into cells with siPORT-NeoFX (Invitrogen, AM4511) and incubated for 24 hours. Cells were washed twice with PBS and lysed with 700 µl lysis buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 0.3% NP40), including 50 U RNaseOUT (Invitrogen, 10777-019) and proteinase inhibitor (Sigma-Aldrich, S8820). Cells were scraped and incubated on ice for 5 min; after centrifugation at 10000 × g (4°C) for 5 min, supernatants were collected.

Streptavidin purification

Streptavidin-coated magnetic beads (Invitrogen, 11205D) were incubated for 2 hours at 4°C with blocking buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 100 mM KCl, 0.3% NP40, 1 mg/ml yeast tRNA, 1 mg/ml BSA). After blocking, cell lysate was added to the magnetic beads, and incubated for 4 hours at 4°C. Four hours later, the beads were washed 5 times with lysis buffer, and RNA was isolated from the beads by the method described previously.36

MicroRNA-mimic transfection

2.5 × 105 IB3-1/S9 cells were plated on a 6-well plate, and incubated in LHC-8 media for 24 hours. Twenty-four hours later, 10 nM of miR-155 mimic or control mimic was transfected into cells with siPORT-NeoFX; 48 hours after that, RNA was isolated from cells by the method described previously.36

RNA extraction and labeling

RNA quality and quantity was evaluated using the RNA6000 Agilent Bioanalyzer chip (Agilent, Santa Clara, CA). Five hundred ng total RNA was labeled according to the manufacturer's instructions using the Illumina® TotalPrep™ RNA amplification kit (Thermo Fisher Scientific, Waltham, MA) to produce biotinylated RNA via first- and second-strand reverse transcription steps and a single in vitro transcription amplification incorporating biotin-labeled nucleotides.

mRNA expression profiling

mRNA expression profiling for biotinylated microRNA transfection and microRNA-155 mimic transfection were done with HumanHT-12 v4 Expression BeadChip Kit (Illumina, BD-103-0204). Arrays were hybridized with 750 ng biotinylated RNA overnight at 58°C. Following standard post-hybridization rinses, blocking and staining with streptavidin-conjugated Cy3, the fluorescent hybridization signal was quantified on an Illumina iScan scanner at a resolution of 0.53 micron and analyzed using Illumina GenomeStudio Software (V2011.1, Gene expression module 1.9.0.). The raw signals were normalized by Z-transformation and differences of Z-scores of test samples and controls samples were calculated for all mRNA signals. mRNAs which were detected more in biotinylated miR-155 treatment and less after miR-155 mimic transfection were selected for further investigation.

mRNA, miRNA and protein expression levels

Total RNA was isolated from the IB3-1 and IB3-1/S9 cells using the miRVana isolation kit (Life Technologies, AM1560). Real-time quantifications of individual mRNA and miRNAs were performed with specific TaqMan gene expression or microRNA assay (Life Technologies). Real-time PCR data were normalized to the endogenous control β-actin mRNA (for mRNA expression, #Hs01060665_g1) or RNU48 (for miRNA expression, #1006) and filtered for Ct values >35. To determine protein expression levels, 2.5 × 105 cells were plated in a 6-well plate. After 24 hours, (70-80% confluent) IB3-1/S9 cells were transfected with 50 nM of pre-miR-155 (Ambion, #AM12601) or pre-miR negative control #1 (Ambion, #AM17110) with siPORT-NeoFX and incubated for 48 hours. Alternatively, 24 hours after plating IB3-1 cell, 150 nM of anti-miR-155 (Ambion, #AM12601) or anti-miR negative control #1 (Ambion, #AM17010) was transfected with siPORT-NeoFX and incubated for 48 hours. Western blotting was done and membranes were probed with primary antibody for RPTOR (Millipore, 09217, 1:1000). GAPDH was used as a loading control. Quantification of protein expression was performed with ImageJ software. The primary bronchial epithelial cells were obtained from lung brush biopsies of CF patients and mRNA expression levels were analyzed by using Illumina bead arrays as described earlier.37

Statistical analysis for RT-qPCR and Western blot

Ct values obtained by reverse transcription (RT) followed by real-time, quantitative (q)PCR analysis for mRNA and miRNAs were analyzed by ΔΔCt method, following analysis guidelines of Applied Biosystems. Average and standard deviation of ΔΔCt were calculated (AvgΔΔCt and StdevΔΔCt) and relative expression is expressed by 2-AvgΔΔCt. The error bars span 2-AvgΔΔCt±StdevΔΔCt. Statistical difference of AvgΔΔCt between control and test conditions was evaluated by 2-tailed t-test, and considered as significant when p-value was less than 0.05.

The intensity of bands from Western blot films was quantified by ImageJ software and test protein signals were normalized to loading control protein signals. Standard errors of the mean (SEM) were calculated from the normalized divided value. Experiments were repeated at least 3 times, and representative blots are shown.

Plasmids for luciferase assay

Plasmid for luciferase assay was obtained from pMIR-REPORT™ miRNA Expression Reporter Vector System (ThermoFisher Scientific, AM5795). 53 nt-long sequence containing the predicted sites of WT and mutant of 3′- UTR sequence of RPTOR were inserted between SpeI and HindIII restriction sites of pMIR-Reporter vector. Exact insert sequences are: WT 5′-actagtccACCTCACTTTATTTCCATGTAATCAGAGCATTAGCTGCcaagctt-3′ and mutant 5′-actagtccACCTCACTTTATTTCCATGTAATCAGACGTAAAGCTGCcaagctt-3′. The vector without any inserts was used as positive control as well (designated as Luciferase only).

Luciferase assay

Luciferase assay was performed with pMIR-REPORT™ miRNA Expression Reporter Vector System (ThermoFisher Scientific, AM5795). IB3-1/S9 cells (2.5 × 105) were plated in 6-well plates 24 hours before transfection. The cells were transfected with 250 ng/ml of pMIR-Reporter, pMIR-Reporter-WT 3′-UTR, or pMIR-Reporter-mutant 3′-UTR. To control for transfection efficiency, pMIR-Reporter-β-galactosidase control vector was co-transfected. 50 nM of pre-miR-155 or pre-miR negative control was also transfected simultaneously. Transfection was performed using siPORT-NeoFX (Invitrogen). After incubating for 24 hours cells were lysed with Tropix lysis solution and luciferase activity in the cell lysates were assayed using Dual-Light Luciferase and β-Galactosidase Reporter Gene Assay System (Life Technologies, T1003). Luminescence was measured with BioTek™ Synergy™ H1 Hybrid Multi-Mode Monochromator Fluorescence Microplate Readers (BioTek Instruments, Inc.). The relative luciferase activity was normalized.

Treatment with rapamycin

IB3-1/S9 cells (2.5 ×105) were treated with 50 nM rapamycin (LC Laboratories, R-5000) or equal volume of DMSO (0.005 vol%) and incubated for 48 hours. Cell lysate was collected with RIPA buffer containing protease inhibitor (Sigma-Aldrich, #S8820-20TAB) and phosphatase inhibitor cocktail (Sigma-Aldrich, #P5726-1ML). The effect of rapamycin was confirmed by measuring total and phosphorylated mTOR protein expression by western blot as described previously (mTOR (7C10) Rabbit mAb #2983, phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb #5536, Cell Signaling, 1:1000). CTGF protein expression was measured by protein gel blot analysis (CTGF, Santa Cruz biotechnology, sc-14939, 1:2000). The levels of mRNAs encoding selected inflammatory proteins was measured by RT-qPCR analysis as described above, by using TaqMan gene expression assay, and relative expression was calculated as described above by using β-actin mRNA as a control [#Hs00174097_m1 (IL-1β), # Hs00985639_m1 (IL-6), #Hs00174103_m1 (IL8), #Hs00961622_m1 (IL-10), #Hs01026927_g1 (CTGF), #Hs00998133_m1 (TGF-β), # Hs00989291_m1 (IFN-γ), #Hs01060665_g1 (β-actin), Life Technologies].

Treatment with TGF-β signaling pathway inhibitor

IB3-1 cells (2.5 × 105) were treated with 2 µM of SB431542 (Calbiochem, #616461) or equal volume of DMSO (0.02 vol%), and incubated for 48 hours. Cell lysate was collected with RIPA buffer containing protease inhibitor (Sigma-Aldrich, #S8820-20TAB) and phosphatase inhibitor cocktail (Sigma-Aldrich, #P5726-1ML). The effect of SB431542 was confirmed by measuring total and phosphorylated SMAD2 protein expression by western blot as described above (phospho-SMAD2, Cell Signaling #3101S, 1:800, and total SMAD2/3, Cell Signaling #5678, 1:2000). CTGF protein expression was measured by Western blot as described above.

Disclaimer

The views expressed are those of the authors and do not reflect the official policy or position of the Uniformed Services University of the Health Sciences, the Department of the Defense, or the United States government.

Disclosure of potential confllicts of interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by Cystic Fibrosis Foundation Research Grant and USUHS Intramural funds to RB. YZ, EL, KGB, and MG were supported by the NIA-IRP, NIH.

References

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