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Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 RESEARCH ARTICLE Open Access Overexpression of miR-9 in mast cells is associated with invasive behavior and spontaneous metastasis Joelle M Fenger1, Misty D Bear2, Stefano Volinia3, Tzu-Yin Lin4, Bonnie K Harrington2, Cheryl A London1,2 and William C Kisseberth1* Abstract Background: While microRNA (miRNA) expression is known to be altered in a variety of human malignancies contributing to cancer development and progression, the potential role of miRNA dysregulation in malignant mast cell disease has not been previously explored. The purpose of this study was to investigate the potential contribution of miRNA dysregulation to the biology of canine mast cell tumors (MCTs), a well-established spontaneous model of malignant mast cell disease. Methods: We evaluated the miRNA expression profiles from biologically low-grade and biologically high-grade primary canine MCTs using real-time PCR-based TaqMan Low Density miRNA Arrays and performed real-time PCR to evaluate miR-9 expression in primary canine MCTs, malignant mast cell lines, and normal bone marrow-derived mast cells (BMMCs). Mouse mast cell lines and BMMCs were transduced with empty or pre-miR-9 expressing lentiviral constructs and cell proliferation, caspase 3/7 activity, and invasion were assessed. Transcriptional profiling of cells overexpressing miR-9 was performed using Affymetrix GeneChip Mouse Gene 2.0 ST arrays and real-time PCR was performed to validate changes in mRNA expression. Results: Our data demonstrate that unique miRNA expression profiles correlate with the biological behavior of primary canine MCTs and that miR-9 expression is increased in biologically high grade canine MCTs and malignant cell lines compared to biologically low grade tumors and normal canine BMMCs. In transformed mouse malignant mast cell lines expressing either wild-type (C57) or activating (P815) KIT mutations and mouse BMMCs, miR-9 overexpression significantly enhanced invasion but had no effect on cell proliferation or apoptosis. Transcriptional profiling of normal mouse BMMCs and P815 cells possessing enforced miR-9 expression demonstrated dysregulation of several genes, including upregulation of CMA1, a protease involved in activation of matrix metalloproteases and extracellular matrix remodeling. Conclusions: Our findings demonstrate that unique miRNA expression profiles correlate with the biological behavior of canine MCTs. Furthermore, dysregulation of miR-9 is associated with MCT metastasis potentially through the induction of an invasive phenotype, identifying a potentially novel pathway for therapeutic intervention. Keywords: Mast cell, microRNA, miR-9 * Correspondence: kisseberth.2@osu.edu 1 Department of Veterinary Clinical Sciences, Columbus, USA Full list of author information is available at the end of the article © 2014 Fenger et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 Background Mast cell-associated malignancies are important diseases in both humans and dogs [1,2] and are characterized by activating mutations in KIT in both species. More than 90% of human patients with systemic mastocytosis carry the D816V mutation in KIT [3] which results in constitutive activation of KIT signaling and plays a major role in the proliferative phenotype. A functionally identical mutation (D814V) is found in transformed mast cell lines from rodents [4,5]. Similarly, approximately 30% of dogs with high-grade cutaneous mast cell tumors (MCTs) possess activating internal tandem duplications (ITDs) in the KIT juxtamembrane (JM) domain [6,7]. More recently, activating mutations in the extracellular domain of KIT (exons 8 and 9) have also been identified in a proportion of canine MCTs [8]. While the role of KIT dysfunction in mast cell neoplasia has been well described, little is known regarding additional molecular mechanisms that may contribute to invasion and metastasis of malignant mast cells. The expression of matrix metalloproteinases (MMPs), a family of enzymes involved in the degradation and remodeling of extracellular matrix, has been implicated in the neoplastic transformation of mast cells. Normal canine bone marrow-derived mast cells (BMMCs) produce large quantities of inactive and active MMP9 in response to various stimuli while releasing little detectable MMP2 [9]. Neoplastic mast cells are known to produce both MMP2 and MMP9 [10] suggesting that the ability to produce MMP2 may be a feature acquired by malignant mast cells. Furthermore, high-grade MCTs express significantly higher levels of MMP9 in proactive and active forms, which has been proposed to be associated with the high degree of malignant behavior of these tumors [10,11]. More recently, characterization of the proteome of primary canine low-grade MCTs and aggressive, high-grade MCTs identified differentially expressed proteins between the two groups [12]. Several stress response proteins (HSPA9, TCP1A, TCP1E) and cytoskeletal proteins associated with actin remodeling and cell migration (WDR1) were significantly up-regulated in high-grade MCTs. MicroRNAs (miRNAs) are highly conserved, noncoding RNAs that serve as important regulators of gene expression. It is well established that miRNA expression is altered in many human malignancies and that miRNAs function as tumor suppressor genes or oncogenes through dysregulation of target genes [13]. Currently there is limited information regarding the potential role of miRNA dysregulation in malignant mast cell disease. Several miRNAs appear to play an important role in normal murine mast cell differentiation [14] and following activation of murine mast cells, up-regulation of the miR-221-222 family influences cell-cycle checkpoints, in Page 2 of 16 part by targeting p27Kip1 [15]. Basal levels of miR-221 contribute to the regulation of the cell cycle in resting mast cells. However, its effects are activation-dependent and in response to mast cell stimulation; miR-221 regulates degranulation, cytokine production, and cell adherence [16]. More recent studies have demonstrated roles for miR-539 and miR-381 in mediating a novel regulatory pathway between KIT and microphthalmia-associated transcription factor in normal and malignant mast cells [17]. The purpose of this study was to investigate the potential role of miRNA dysregulation in the biologic behavior of primary canine MCTs. We found that unique miRNA expression profiles correlate with the biological behavior of primary canine MCTs and that miR-9 was significantly overexpressed in aggressive MCTs compared to benign MCTs. Furthermore, enforced miR-9 expression in murine mastocytoma cell lines and normal murine BMMCs with low basal levels of miR-9 enhanced invasion and induced the expression of several target genes associated with Table 1 Primers for quantitative reverse transcriptase polymerase chain reaction Primers Primer sequences Mouse Cma1 292F 5’-GAA GAC ACG TGG CAG AAG CTT GAG-3’ Mouse Cma1 521R 5’-GTG TCG GAG GCT GGC TCA TTC ACG-3’ Mouse Hspe F479 5’-GCT CAG TGG ACA TGC TCT ACA G-3’ Mouse Hspe R697 5’-GCA ACC CAT CGA TGA GAA TGT G-3’ Mouse Ifitm3 115F 5’-GCT TCT GTC AGA ACT ACT GTG-3’ Mouse Ifitm3 339R 5’-GAG GAC CAA GGT GCT GAT GTT CAG-3’ Mouse Mlana 125F 5’-GCT GCT GGT ACT GTA GAA GAC G-3’ Mouse Mlana 322R 5’-GTG AAG AGA GCT TCT CAT AGG CAG-3’ Mouse Pdzk1ip1 F520 5’-GTT CTG GCT GAT GAT CAC TTG ATT G-3’ Mouse Pdzk1ip1 R769 5’-GAT AGA AGC CAT AGC CAT TGC TG-3’ Mouse SerpinF1 712F 5’-GTG AGA GTC CCC ATG ATG TCA G-3’ Mouse SerpinF1 910R 5’-GTT CTC GGT CGA TGT CAT GAA TG-3’ Mouse Tlr7 F2284 5’-GTC ATT CAG AAG ACT AGC TTC CCA G-3’ Mouse Tlr7 R2441 5’-GTC ACA TCA GTG GCC AGG TAT G-3’ Mouse Cd200r1 659F 5’-GTA ACC AAT CTC TGT CCA TAG-3’ Mouse Cd200r1 902R 5’-GTC ACA GTA TCA TAG AGT GGA TTG-3’ Mouse Cd200r4 312F 5’-GCC TCC ACA CCT GAC CAC AG-3’ Mouse Cd200r4 532R 5’-GTC CAA GAG ATC TGT GCA GCA G-3’ Mouse Perp F108 5’-GCA GTC TAG CAA CCA CAT CCA G-3’ Mouse Perp R267 5’-GCA CAG GAT GAT AAA GCC ACA G-3’ Mouse Slpi F142 5’-GAG AAG CCA CAA TGC CGT ACT G-3’ Mouse Slpi R378 5’-GAC TTT CCC ACA TAT ACC CTC ACA G-3’ Mouse Pparg F682 5’-GAT ATC GAC CAG CTG AAC CCA G-3’ Mouse Pparg R983 5’-GCA TAC TCT GTG ATC TCT TGC ACG-3’ 18S V2F 5’-AAA TCC TTT AAC GAG GAT CCA TT-3’ 18S V2R 5’-AAT ATA CGC TAT TGG AGC TGG A-3’ Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 metastasis, including chymase (CMA1) and heparinase (HSPE). These data suggest that miR-9 overexpression may contribute to the invasive phenotype of malignant mast cells thereby providing a potentially novel pathway for therapeutic intervention in malignant mast cell disease. Methods Cell lines, primary cell cultures, primary tumor samples Mouse P815 (D814V KIT mutation) and C57 (wild-type KIT) cell lines were provided by Dr. Stephen Galli (Stanford University). The canine BR (activating point mutation L575P in the JM domain of KIT) and C2 (KIT ITD mutation in the JM domain) cell lines were provided by Dr. Warren Gold (Cardiovascular Research Institute, University of California- San Francisco). Cell lines were maintained in RPMI 1640 (Gibco® Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco® Life Technologies) and antibiotics (Gibco® Life Technologies). Mouse BMMCs were generated from bone marrow from C57/B6 wild-type mice as previously described [9]. Canine BMMCs were generated from 2 dogs and maintained in Stemline (SigmaAldrich, St. Louis, MO, USA) medium supplemented with recombinant canine stem cell factor (R & D Systems, Minneapolis, MN, USA) as previously described [18]. Protocols for collection of murine bone marrow and canine bone marrow were approved by the Ohio State University Page 3 of 16 (OSU) Institutional Care and Use Committee (IACUC), protocols 2009A0204 and 2010A0015, respectively. Canine MCTs were obtained from 24 different affected dogs presented to the OSU Veterinary Medical Center and University of California-Davis (UCD) Veterinary Teaching Hospital. Tumor sample collections were performed in accordance with established hospital protocols and approved by respective IACUC at both OSU and UCD. Clinical outcome data, including sex, breed, primary tumor location, recurrence and metastasis, histopathologic grade, mitotic index, and outcome was available for all dogs (see Additional file 1). Tumors obtained from dogs that were adequately controlled with surgery alone and did not develop or die from metastatic mast cell disease were considered biologically low-grade tumors (benign). Tumors from dogs that developed aggressive, metastatic mast cell disease which resulted in their death were classified as biologically high-grade tumors. Quantitative reverse-transcription-PCR profiling of mature miRNA expression in MCT biopsies Total RNA was isolated by the Trizol method (Invitrogen, Carlsbad, CA, USA) and heparinase treated as described [19]. Primary MCT miRNA expression profiling was performed at the OSU Nucleic Acid Shared Resource using the TaqMan Array Human miRNA Panel (Human A Cards, v.2, Applied Biosystems, Foster City, CA, USA) as Figure 1 MiRNA expression in primary canine MCTs is associated with biological behavior. Primary canine MCTs were obtained from dogs diagnosed with benign tumors (n = 12) or biologically high grade metastatic tumors (n = 12). Real-time PCR profiling was performed using Applied Biosystems Human TaqMan Low Density miRNA Arrays to assess mature miRNA expression in primary tumors. Unsupervised hierarchical cluster analysis separated samples into two groups based on biological behavior and demonstrate unique miRNA expression profiles associated with biologically low-grade (L) tumors or high-grade (H) tumors (P < 0.05). (*) indicates primary tumor sample from a dog with a benign mast cell tumor that clustered with the biologically high grade MCT group. Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 Page 4 of 16 described previously [20]. This panel assays the expression of 377 human miRNAs, 151 of whose mature sequences are 100% conserved between human and dog (Sanger miRBase v.12). Raw data analysis, normalizer selection and statistical analysis were performed using the real-time PCR analysis software Statminer (Integromics, Madison, WI, USA). The snRNA U6 was confirmed to be stably expressed in our sample set and the mean used as the normalizer value. Relative gene expression was calculated using the comparative threshold cycle method [21]. Gene expression heat maps were generated using Treeview PCbased software [22]. RNA isolation and quantitative real-time PCR RNA was extracted from cell lines using TRIzol (Invitrogen) and real-time PCR was performed using the Applied Biosystems StepOne Plus Detection System. MiR-9 is highly conserved and shares 100% homology between dogs, humans, and mice. Mature miR-9 expression was performed using Taqman miRNA assays (Applied Biosystems). 50 ng total RNA was converted to firststrand cDNA with miRNA-specific primers, followed by real-time PCR with TaqMan probes. All samples were normalized to U6 snRNA. Real-time PCR was performed to validate changes in mRNA expression for selected genes affected by miR-9 over expression. cDNA was made from 1 μg of total RNA using Superscript III (Invitrogen). CMA1, HSPE, IFITM3, MLANA, PERP, PPARG, PDZK1IP1, SERPINF1, SLPI, TLR7, CD200R1, CD200R4 and 18S transcripts were detected using Fast SYBR green PCR master mix (Applied Biosystems) according to the manufacturer’s Table 2 MiRNA signature associated with biologically high-grade MCTs miRNA Fold-change p-value miRNA Fold-change Gene expression Gene expression High vs low grade MCT High vs low grade MCT p-value Upregulated miRNAs hsa-miR-301b 4.2 0.00022 hsa-miR-520b 1.8 1.8 hsa-miR-454 2.4 0.00032 hsa-miR-216b 4.6 0.023 hsa-miR-9 3.2 0.0010 hsa-miR-302b 3.2 0.024 hsa-miR-147 3.9 0.0017 hsa-miR-106b 1.6 0.026 hsa-miR-138 2.5 0.0022 hsa-miR-618 3.0 0.027 hsa-miR-330-5p 3.1 0.0027 hsa-miR-518f 3.2 0.029 hsa-miR-187 5.1 0.0029 hsa-miR-182 2.8 0.030 hsa-miR-106a 2.1 0.0044 hsa-miR-142-5p 1.7 0.031 hsa-miR-636 2.7 0.0052 hsa-miR-301a 2.8 0.032 hsa-miR-17 2.0 0.0057 hsa-miR-217 3.9 0.033 hsa-miR-449b 3.2 0.0069 hsa-miR-652 2.0 0.039 hsa-miR-130b 2.2 0.0082 hsa-miR-186 1.5 0.039 hsa-miR-192 2.5 0.0095 hsa-miR-19a 1.8 0.040 hsa-miR-448 3.1 0.010 hsa-miR-872 1.5 0.041 hsa-miR-425 3.0 0.011 hsa-miR-148b 1.8 0.043 hsa-miR-193a-3p 2.6 0.011 hsa-miR-451 2.4 0.044 hsa-miR-18b 2.2 0.014 hsa-miR-423-5p 1.7 0.048 hsa-miR-93 2.1 0.014 hsa-miR-191 1.5 0.049 hsa-miR-548b-5p 2.3 0.015 Downregulated miRNAs hsa-miR-25 2.1 0.015 hsa-miR-885-5p -4.2 0.00011 hsa-miR-324-3p 2.3 0.017 hsa-miR-874 -5.8 0.00018 hsa-miR-326 2.6 0.017 hsa-miR-486-3p -4.6 0.00040 hsa-miR-18a 3.1 0.017 hsa-miR-299-5p -4.2 0.0020 hsa-miR-20b 2.0 0.017 hsa-miR-488 -3.9 0.0063 hsa-miR-194 2.8 0.019 hsa-miR-200a -5.5 0.034 hsa-miR-372 2.4 0.019 hsa-miR-412 -2.8 0.035 Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 Page 5 of 16 protocol; primer sets are detailed in Table 1. Normalization was performed relative to 18S rRNA. All reactions were performed in triplicate and included notemplate controls for each gene. Relative gene expression for all real-time PCR data was calculated using the comparative threshold cycle method [21]. Experiments were repeated 3 times using samples in triplicate. MiR-9 lentivirus infection Lentiviral constructs were purchased from Systems Biosciences (Mountain View, CA, USA). Packaging of the lentiviral constructs was performed using the pPACKH1 Lentivector Packaging KIT (catalog no. LV500A-1) according to the manufacturer’s instructions. P815 and C57 mouse mastocytoma cells and mouse BMMCs (105 cells) were transduced with empty lentivirus (catalog no. CD511B-1) or pre-miR-9-3 lentivirus (catalog no. PMIRH9-3PA-1). FACS-mediated cell sorting based on GFP expression was performed 72 hours post-transduction and miR-9 expression was evaluated by real-time PCR (Applied Biosystems). Transcriptional profiling of cells transduced with miR-9 lentivirus RNA was extracted from mouse BMMCs and P815 cells transduced with empty lentivirus or pre-miR-9-3 lentivirus from three separate transduction experiments using TRIzol (Invitrogen). A secondary RNA cleanup step was performed using QIAGEN RNeasy Total RNA isolation kit (QIAGEN GmbH, Hilden, Germany) and RNA integrity was assessed using RNA 6000 Nano LabChip® Kits on the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). RNA was labeled Matrigel invasion assay To assess the effect of miR-9 expression on invasion, cell culture inserts (8-μm pore size; Falcon) were coated with 100 μL of Matrigel (BD Bioscience, San Jose, CA, USA) to form a thin continuous layer and allowed to solidify at 37°C for 1 hour. P815 and C57 cell lines, and mouse BMMCs (5 × 105/mL) transduced with control lentivirus or pre-miR-9-3 lentivirus were prepared in serum-free medium and seeded into each insert (upper chamber) and media containing 10% fetal bovine serum was placed in the lower chamber. The cells were incubated for 24 hours to permit invasion through the Matrigel layer. Cells remaining on the upper surface of the insert membrane were wiped away using a cotton swab, and cells that had migrated to the lower surface were stained with crystal violet and counted in ten independent 20× high powered fields for each sample. Experiments were repeated 3 times using samples in triplicate. B 0.018 MiR-9 Gene Expression, 2- CT MiR-9 Gene Expression, 2- CT A 0.020 with Cy3 using RNA ligase and hybridized to GeneChip® Mouse Gene 2.0 ST Arrays (Affymetrix, Santa Clara, CA, USA). Ratios of signals were calculated and transcripts that were up-regulated or down-regulated by at least 2-fold were identified (p < 0.05). Data analysis, statistical analysis, and generation of gene expression heat maps were performed using Affymetrix® Transcriptome Analysis Console (TAC) Software. Prediction of miR-9 binding to the 3’-UTR of genes down-regulated by miR-9 was performed with computer-aided algorithms obtained from TargetScan (http://www.targetscan.org), PicTar (http://pictar.mdc-berlin.de), miRanda (http://www.microrna.org), and miRWalk (http://www.umm.uni-heidelberg. de/apps/zmf/mirwalk). 0.016 0.014 0.012 0.010 0.008 0.006 * 0.004 0.002 0.000 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 0.000 Low Grade MCTs High Grade MCTs cBMMC BR Canine C2 mBMMC P815 Mouse C57 Figure 2 MiR-9 is highly expressed in biologically high grade canine MCTs and malignant mast cell lines. (A) Real-time PCR evaluating mature miR-9 expression in primary canine MCTs demonstrated that the mean expression of miR-9 was 3.2-fold higher in aggressive, high grade MCTs compared to benign MCTs (p = 0.001). (*) indicates primary tumor sample from a dog with a low-grade mast cell tumor that expressed high levels of miR-9 but had lymph node metastasis at the time of surgery. (B) Malignant canine BR and C2 mast cells, normal canine and mouse BMMCs, and malignant mouse C57 and P815 cells were cultured and real-time PCR was performed to assess miR-9 expression levels. Three independent experiments were performed and all reactions were performed in triplicate. The experiments were repeated 3 times in the cell lines and twice for normal cBMMCs. Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 Page 6 of 16 Evaluation of proliferation and apoptosis Cell proliferation was calculated as a percentage of untransduced control cells. Caspase-3/7 activity was determined using the SensoLyte® Homogeneous AMC Caspase- 3/7 Assay KIT (Anaspec Inc, San Jose, CA, USA) as previously described [24]. P815 and C57 cells (5.0 × 104) transduced with either empty lentivirus or pre-miR-9-3 lentivirus were plated for 24 and 48 hours in 96-well plates prior to analysis. Fluorescence was measured on a SpectraMax microplate reader (Molecular Devices). Levels of caspase Changes in cell proliferation were assessed using the CyQUANT® Cell Proliferation Assay KIT (Molecular Probes, Eugene, OR, USA) as previously described [23]. P815 and C57 cells (15 × 104) transduced with control lentivirus or pre-miR-9-3 lentivirus were seeded in 96-well plates for 24, 48, and 72 hours prior to analysis. Nontransduced P815 and C57 cells served as negative control wells. Fluorescence was measured using a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). * 0.025 B Mean Number Invaded Cells/hpf 0.035 P815 (D814V) KIT) C57 (WT KIT) * 0.030 0.025 0.020 0.020 0.015 0.015 0.010 0.010 0.005 0.005 0.000 0.000 WT C EV miR-9 WT EV % Cells Surviving (% Control) EV miR9 100 80 60 40 20 * 9 8 25 7 20 6 5 15 4 10 3 2 5 1 0 0 EV miR-9 WT EV miR-9 400 EV miR9 300 200 100 0 24h 48h 72h 24h 3000 P815 (D814V) KIT) 48h P815 (D814V) KIT) 2500 100 Flourescence (RFU) % Cells Surviving (% Control) 30 500 C57 (WT KIT) 0 120 * WT D 120 C57 (WT KIT) 10 P815 (D814V) KIT) 35 C57 (WT KIT) miR-9 Flourescence (RFU) MiR-9 Gene Expression, 2- CT A 0.030 80 60 40 20 2000 1500 1000 500 0 0 24h 48h 72h 24h 48h Figure 3 Overexpression of miR-9 enhances invasion of malignant mast cells and has no effect on cell proliferation or apoptosis. (A) Mouse P815 and C57 mast cells transduced with pre-miR-9-3 lentivirus or empty vector control were sorted to greater than 95% purity based on GFP expression. MiR-9 levels were assessed by real-time PCR in wild-type, empty vector, and miR-9 expressing cells (*p < 0.05). Three independent experiments were performed and all reactions were performed in triplicate. (B) Mouse P185 and C57 mast cells transduced with either empty vector or pre-miR-9-3 lentivirus were transferred onto cell culture inserts coated with Matrigel® for 24 hrs. After incubation, membranes were stained and cells that had invaded the membrane were counted in ten independent 20x hpf for each sample. Three independent experiments were performed and all assays were performed in triplicate wells (*p < 0.05). (C) Mouse P185 and C57 mast cells were transduced with either empty vector or pre-miR-9-3 lentivirus vector and cell proliferation was analyzed at 24, 48, and 72 hours using the CyQUANT method. Nontransduced P815 and C57 cells served as non-treated controls. Three independent experiments were performed and all samples were seeded in triplicate wells. Values are reported as percentage of untransduced control cells. (D) Mouse P185 and C57 mast cells transduced with either empty vector or pre-miR-9-3 lentivirus were assessed for apoptosis at 24 and 48 hours by measuring active caspase-3/7 using the SensoLyte® Homogeneous AMC Caspase-3/7 Assay kit. Relative fluorescence units are reported after subtraction of fluorescence levels of wells with medium only. Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 Page 7 of 16 3/7 activity were reported after subtraction of fluorescence levels of wells with medium only. Statistical analysis Statistical analysis relative to miRNA expression data was performed with Statminer software (Integromics) and p-values of <0.05 were considered statistically significant. Statistical analysis relative to mRNA expression data was performed using Affymetrix® Transcriptome Analysis Console (TAC) Software. Differential gene expression was determined by one-way ANOVA comparison test and p-values of <0.05 were considered statistically significant. All experiments with the exception of those involving canine BMMCs were performed in triplicate and repeated 3 times. Experiments using canine BMMCs were performed in triplicate, but repeated only twice because of limited cell numbers. Data were presented as mean plus or minus standard deviation. The difference between two group means was analyzed using the Students t-test and a one-way analysis of variance (ANOVA) was performed for multiple variable comparisons. P-values of <0.05 were considered significant. Results MiRNA expression in primary canine MCTs is associated with biological behavior To investigate the role of miRNA dysregulation in the biologic behavior of mast cell disease, global miRNA expression in primary canine MCTs obtained from 24 dogs miR-9 is overexpressed in biologically high-grade canine MCTs The miRNA array performed above identified miR-9 as overexpressed in MCTs that metastasized and resulted in death of affected dogs. This finding was confirmed by real-time PCR in which a 3.2-fold increase in miR-9 expression was identified in biologically aggressive MCTs as compared to benign MCTs (Figure 2A). Furthermore, miR-9 expression correlates with tumor grade and metastatic status in human breast cancer, providing further support for the idea that altered miR-9 expression may be an important regulator of aggressive biological behavior in MCTs (33). Interestingly, one of the primary tumor B 0.018 mBMMCs * 0.016 0.014 0.012 0.010 0.008 0.006 0.004 0.002 0.000 WT EV miR-9 Mean Number Invaded Cells/hpf MiR-9 Gene Expression, 2- CT A diagnosed with benign tumors (n = 12) or with biologically high-grade tumors (n = 12) was evaluated using realtime PCR-based TaqMan Low Density miRNA Arrays (Applied Biosystems). An unsupervised hierarchial cluster analysis of all primary MCTs readily separated tumors into groups based on biological behavior with aggressive, highly metastatic MCTs clustering together and clinically benign MCTs clustering together separately (Figure 1). We identified 45 miRNAs that had significantly higher expression in biologically highgrade MCTs compared to biologically low-grade MCTs, while 7 miRNAs had lower expression (Table 2). These data demonstrate that biologically high-grade and lowgrade canine MCTs possess distinct miRNA expression signatures. 25 mBMMCs * 20 15 10 5 0 EV miR-9 Figure 4 Overexpression of miR-9 enhances invasion in normal mouse bone marrow-derived mast cells. (A) Normal mBMMCs transduced with pre-miR-9-3 lentivirus or empty vector control were sorted to greater than 95% purity based on GFP expression. MiR-9 levels were assessed by real-time PCR (*p < 0.05). Three independent experiments were performed and all reactions were performed in triplicate. (B) mBMMCs transduced with either empty vector or pre-miR-9-3 lentivirus were transferred onto cell culture inserts coated with Matrigel® for 24 hrs. After incubation, cells remaining on the upper surface of the insert membrane were wiped away using a cotton swab, and cells that had migrated to the lower surface were stained with crystal violet and counted in ten independent 20x hpf for each sample. Three independent experiments were performed and all samples were performed in triplicate wells (*p < 0.05). Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 Page 8 of 16 Figure 5 (See legend on next page.) miR9 miR9 miR9 2.02 EV EV EV mBMMC 13.23 Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 Page 9 of 16 (See figure on previous page.) Figure 5 Overexpression of miR-9 in normal mouse bone marrow-derived mast cells significantly alters gene expression. Normal mBMMCs transduced with pre-miR-9-3 lentivirus or empty vector control were sorted based on GFP expression. RNA was harvested from mouse BMMCs transduced with empty vector or pre-miR-9-3 lentivirus from three separate transduction experiments. Transcriptional profiling was performed using Affymetrix GeneChip® Mouse Gene 2.0 ST Arrays. Hierarchical clustering was performed for 450 genes differentially expressed (p < 0.05) in mBMMCs expressing either empty vector (EV) or miR-9 (miR9) as determined by one-way ANOVA comparison test (p < 0.05). Mean centered signal intensities of gene-expression are depicted by the log2 of the ratio of the signals against the average signal for each comparison. Color areas indicate relative expression of each gene after log2 transformation with respect to the gene median expression (red above, green below, and black equal to the mean). samples collected from a dog with a biologically lowgrade MCT expressed high levels of miR-9 and the unsupervised hierarchial clustering of all 24 MCTs demonstrated that this dog’s tumor clustered with the biologically high-grade tumors (Figure 1). Clinical data was subsequently reviewed for all dogs and it was determined that this dog had histopathologically confirmed evidence of metastatic mast cells present in a regional lymph node surgically excised at the time of primary tumor removal. Additionally, one high-grade MCT clustered with the low-grade tumors, however, this may have been due, in part, to variations in stroma/ inflammatory cells within the primary tumor specimen or baseline necrosis within the tumor that influenced the proportion of tumor cells. Taken together, these findings suggest a correlation between miR-9 expression levels in primary canine MCTs and metastatic behavior. Overexpression of pre-miR-9 enhances invasion of malignant mast cell lines miR-9 expression is up-regulated in canine malignant mast cell lines To investigate whether overexpression of miR-9 in malignant mast cells affected their capacity to proliferate or survive, mouse C57 and P815 cell lines expressing premiR-9-3 lentivirus or empty vector control were cultured for 24, 48, and 72 hrs and the impact on cell proliferation and apoptosis was assessed. No effects of miR-9 on proliferation or apoptosis were observed in either cell line when compared to cells expressing empty vector (Figure 3C and D). Given the potential link between miR-9 expression and biological behavior of MCTs, we next evaluated miR-9 expression in canine (BR and C2) and murine (C57 and P815) mast cell lines and normal canine and murine BMMCs by real-time PCR. As shown in Figure 2B, canine mastocytoma cells exhibited higher levels of miR-9 expression when compared with normal canine BMMCs. In contrast, both mouse C57 and P815 cells and mouse BMMCs demonstrated low basal levels of miR-9. The mouse P815 mastocytoma cell line is a leukemia of mast cell origin, whereas the canine BR and C2 mastocytoma cells are derived from cutaneous tumors. The differences in the biology of these diseases may account for the observed differences in miR-9 expression in canine and murine cell lines. Low miR-9 expression in P815 cells may reflect the fact that these cells represent a true leukemia, in contrast to the BR and C2 cell lines which are derived from cutaneous tumors that would metastasize via the lymphatic system. Given prior work from our laboratory showing that the C2 line exhibits invasive behavior in vitro while the P815 line does not [24], it was possible that miR-9 expression was associated with the invasive behavior of mast cells. To investigate the functional consequences of miR-9 overexpression in malignant mast cell lines, we stably expressed miR-9 in the mouse P815 and C57 cell lines that exhibit low basal levels of this miRNA using an empty or pre-miR-9-3 expressing lentivirus vector. Following transduction, GFP + cells were sorted and miR-9 expression was confirmed by real-time PCR (Figure 3A). The invasive capacity of cells was then evaluated using a standard Matrigel invasion assay after 24 hours of culture. As shown in Figure 3B, enforced expression of miR-9 in C57 and P815 mast cell lines significantly enhanced their invasion compared to cells expressing empty vector. miR-9 has no effect on cell proliferation or caspase-3,7 dependent apoptosis in malignant mast cells miR-9 expression enhances invasion in normal mouse BMMCs To characterize the biological consequences of miR-9 overexpression in normal mast cells, we transduced murine BMMCs with pre-miR-9-3 lentivirus or empty control vector. MiR-9 overexpression in transformed BMMCs was confirmed by quantitative real-time PCR (Figure 4A). To assess the effect of ectopic miR-9 expression on the invasive capacity the BMMCs, a Matrigel invasion assay was again performed. Consistent with findings in the P815 and C57 cell lines, enforced expression of miR-9 in mouse BMMCs significantly enhanced their invasive capacity compared to cells expressing empty vector (Figure 4B). Together, these data suggest that miR-9 promotes an invasive phenotype in mast cells. Fenger et al. BMC Cancer 2014, 14:84 http://www.biomedcentral.com/1471-2407/14/84 Page 10 of 16 Table 3 Gene transcripts altered by miR-9 overexpression in BMMCs Downregulated with miR-9 expression (BMMCs) 1-Sep Ell2 Phgdh 1300014I06Rik Emp1 Pi16 1600029D21Rik Eya2 Plk2 2810025M15Rik Fn1 Plod2 5830428M24Rik Fzd4 Ppap2b A2ld1 Gatm Pparg Akr1c18 Glrp1 Ppic Alox15 Gm10021 Prg2 Amigo2 Gm19524 Prss34 Ankrd22 Gm2663 Psat1, LOC100047252 Ankrd55 Gm6445 Rbp4 Arfip1 Gnpnat1 Reep6 Arg2 Gpc4 Retnla Asb2 Gpt2 Rhoj Asns Grb10 Scd1 Atp1b1 H2-M2 Scn7a Atp8b4 Hal Serpinb9b Awat1 Hdc Sgce BC100530 Hgf Slamf1 Bex1 Il18rap Slc16a1 Bri3bp Il1f9 Slc22a3 C87414 Il6st Slc36a4 Ccdc88c Itk Slc43a3 Ccl17 Klf5 Slc7a1 Ccl24 Klrb1f Slc7a5 Ccl8 Lama5 Slpi Cd209d Lcn2 Snord70 Cd24a LOC100861767 Speer4e, Gm17019 Cd36 LOC100862026 Stfa2 Cdh17 Lrrk2 Stfa2l1 Cdkn2b Mbnl3 Sulf2 Celsr1 Mcpt8 Syne1 Chi3l4 Mgam Taf1d Clec4e Mmp13 Tfrc Colec12 Mrgpra6 Thbs1 Csf3r Niacr1 Tm4sf19 Ctsg Nrg1 Tmem26 Ctsk O3far1 Tnfrsf10b Ctsl Olr1 Tspan7 Dennd2d, 2010016I18Rik Pdlim1 Ube2e2 Dnajc6 Perp Vmn1r129 Ear2, Ear12, Ear3 Pga5 Zbtb10 Egln3 Phf10 Zfp608 Bold indicates predicted miR-9 targets. Microarray analysis identified genes affected by miR-9 To gain insight into possible mechanisms underlying the observed miR-9-dependent invasive behavior of mast cells, we compared the transcriptional profiles of murine BMMCs overexpressing miR-9 to those expressing empty vector and found marked changes in gene expression (Figure 5). In BMMCs overexpressing miR-9, 321 transcripts were significantly up-regulated (>2-fold) and 129 transcripts were significantly down-regulated (Table 3, Table 4). Bioinformatic analysis identified putative miR-9 target sites within the 3’-UTR of 40 gene transcripts that were significantly down-regulated with miR-9 overexpression, suggesting that miR-9 may directly target and regulate expression of these candidate genes (Table 3, bolded). Real time PCR confirmed that one of these genes, peroxisome proliferator-activated receptor δ (PPARG) was down-regulated, a finding consistent with recent studies demonstrating regulation of PPARG by miR-9 through direct targeting of its 3’-UTR [25]. We performed real-time PCR to validate changes in gene expression for several transcripts altered by miR-9 overexpression in BMMCs. Consistent with our microarray results, we found that transcripts for HSPE and TLR7 were significantly up-regulated in BMMCs expressing miR-9, whereas transcripts for PPARG, PERP, and SLPI were significantly down-regulated compared to empty vector controls (Figure 6A). Similar transcriptional profile analysis was performed using malignant mouse P815 cells and we identified 46 transcripts significantly up-regulated (>2-fold) and 48 transcripts significantly down-regulated in the miR-9 expressing P815 cells (Table 5). Bioinformatic analysis identified putative miR-9 target sites within the 3’-UTR of 15 gene transcripts that were significantly downregulated following miR-9 overexpression, suggesting that miR-9 may directly regulate these genes (Table 5, bolded). Real-time PCR demonstrated that expression of SERPINF1 and MLANA transcript was up-regulated in P815 cells overexpressing miR-9, whereas CD200R1 and CD200R4 was down-regulated compared to empty vector controls (Figure 6B). A comparison of the transcriptional profiles both from normal BMMCs and malignant P815 cells overexpressing miR-9 found that most gene transcripts altered by miR-9 were specific to normal or malignant mast cells. We identified 7 gene transcripts (IFITM3, PDZK1IP1, CMA1, MGL1, TMEM223, SLAMF1, CLEC4E) that showed similar changes in expression following miR-9 overexpression in both BMMCs and P815 cells. We performed real-time PCR to validate changes in gene expression for several transcripts altered by miR-9 overexpression, including mast cell chymase (CMA1), interferon-induced transmembrane protein 3 (IFITM3), and PDZK1 interacting protein 1 (PDZK1IP1). Consistent with our microarray results, real-time PCR confirmed that enforced miR-9 expression