Overexpression of miR-9 in mast cells is associated with invasive behavior and spontaneous metastasis

  • Joelle M Fenger3,

    Affiliated with

    • Misty D Bear4,

      Affiliated with

      • Stefano Volinia3,

        Affiliated with

        • Tzu-Yin Lin4,

          Affiliated with

          • Bonnie K Harrington4,

            Affiliated with

            • Cheryl A London3, 4 and

              Affiliated with

              • William C Kisseberth3Email author

                Affiliated with

                BMC Cancer201414:84

                DOI: 10.1186/1471-2407-14-84

                Received: 7 October 2013

                Accepted: 27 January 2014

                Published: 11 February 2014

                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

                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 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 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 (Sigma-Aldrich, 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 (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 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 PC-based 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 first-strand 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 protocol; primer sets are detailed in Table 1. Normalization was performed relative to 18S rRNA. All reactions were performed in triplicate and included no-template 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.
                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’

                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 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).

                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.

                Evaluation of proliferation and apoptosis

                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). 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 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 diagnosed with benign tumors (n = 12) or with biologically high-grade tumors (n = 12) was evaluated using real-time 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 high-grade MCTs compared to biologically low-grade MCTs, while 7 miRNAs had lower expression (Table 2). These data demonstrate that biologically high-grade and low-grade canine MCTs possess distinct miRNA expression signatures.
                http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-84/MediaObjects/12885_2013_4325_Fig1_HTML.jpg
                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.

                Table 2

                MiRNA signature associated with biologically high-grade MCTs

                miRNA

                Fold-change

                p-value

                miRNA

                Fold-change

                p-value

                 

                Gene expression

                  

                Gene expression

                 
                 

                High vs low grade MCT

                  

                High vs low grade MCT

                 

                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

                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 samples collected from a dog with a biologically low-grade 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.
                http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-84/MediaObjects/12885_2013_4325_Fig2_HTML.jpg
                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.

                miR-9 expression is up-regulated in canine malignant mast cell lines

                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.

                Overexpression of pre-miR-9 enhances invasion of malignant mast cell lines

                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.
                http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-84/MediaObjects/12885_2013_4325_Fig3_HTML.jpg
                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.

                miR-9 has no effect on cell proliferation or caspase-3,7 dependent apoptosis in malignant mast cells

                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 pre-miR-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).

                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.
                http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-84/MediaObjects/12885_2013_4325_Fig4_HTML.jpg
                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).

                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).
                http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-84/MediaObjects/12885_2013_4325_Fig5_HTML.jpg
                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).

                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.

                Table 4

                Gene transcripts altered by miR-9 overexpression in BMMCs

                Upregulated with miR-9 expression (BMMCs)

                1810011H11Rik

                Ddx60

                Irg1

                Plxna1

                2310028H24Rik

                Dnaja4

                Itgb5

                Plxnb3

                3110043O21Rik

                Dpep2

                Kcnab3

                Plxnc1

                4930420K17Rik

                Dusp22

                Kcne3

                Ppargc1a

                5033411D12Rik

                E130215H24Rik

                Kctd12

                Ppfibp2

                5430435G22Rik

                E330020D12Rik

                Kctd6

                Ppp1r14c

                6330415B21Rik

                Ednra

                Khdc1a

                Prdx1, LOC100862012

                9030625A04Rik

                Egr1

                Kit

                Prickle1

                9430070O13Rik

                Emx2

                Klf2

                Psd3

                9930111J21Rik2

                Epsti1

                Klk1b1

                Psg23

                A130040M12Rik

                Esco2

                Klk1b11

                Ptafr

                A230098N10Rik

                Esr1

                Klk1b27

                Ptger2

                A430084P05Rik

                Evl

                Klk1b5

                Ptplad2

                A4galt

                F13a1

                Kmo

                Ptpn13

                Abi3

                Fabp5

                Lce6a

                Qpct

                Adamtsl3

                Fabp5, Gm3601

                LOC100038947

                Rasgrp3

                Adrb2

                Fam125b

                LOC100861753

                Rassf4

                AI593442

                Fam55d

                LOC100861977

                Rbm47

                AI607873

                Fam69a

                LOC100862646

                Rin2

                Alcam

                Fcgr4

                Lphn1

                Rnase4, Ang

                Alpk2

                Fkbp1b

                Lrp1

                Rnase6

                Ank

                Fos

                Lrrc16a

                Rnf180

                Ano3

                Fpr2

                Lrrc25

                Rny1

                Aoah

                Galnt10

                Lrrtm1

                Rps6ka2

                Apobec1

                Galntl4

                Ltf

                Rsph9

                Ar

                Gas6

                Ly6i

                Rtp4

                Arhgap20

                Gbp3

                Lyz1

                Ryr3

                Arhgap24

                Gbp4

                Maf

                Scn1b

                Arhgap31

                Gbp5

                Mast4

                Scpep1

                Arl5b

                Gbp8

                Mc1r

                Serpinb8

                Asphd2

                Gbp9

                Mecom

                Siglec1

                Bank1

                Gcet2

                Mgl2

                Sirpb1a

                BC013712

                Gdf15

                Mgll

                Sirpb1b

                Bcl2a1b, Bcl2a1a

                Gdpd1

                Mir15b

                Slc30a2

                Bcl2a1d, Bcl2a1a, Bcl2a1b

                Ggh

                Mir181a-1

                Slc37a2

                Bhlhe41

                Glul

                Mir3095

                Slc39a4

                Bmpr2, Gm20272

                Gm11711, Cd300lh

                Mir3108

                Slc40a1

                Bst1

                Gm12250

                Mir511

                Slc4a11

                Bst2

                Gm14446

                Mir701

                Slc6a12

                C1qb

                Gm15915

                Mlph

                Slc9a9

                C1qc

                Gm1673

                Mmp2

                Slfn5

                C330018A13Rik

                Gm1966

                Mnda, Ifi204

                Smpdl3b

                C5ar1

                Gm20099

                Mpeg1

                Smpx

                Cacnb4

                Gm4759

                Mrgpra9

                Snord14e, Hspa8

                Cadm3

                Gm4951

                Mrgprb2

                St3gal5

                Car8

                Gm5431

                Ms4a4a

                St6galnac3

                Ccl2

                Gm7977

                Ms4a6b

                Stab1

                Ccl4

                Gmpr

                Ms4a6c

                Stfa3

                Ccl7

                Gna14

                Ms4a6d

                Sult1a1

                Ccnd1

                Gp1ba

                Ms4a7

                Syn2

                Ccr1l1

                Gp5

                Msr1

                Syngr1

                Ccr3

                Gpm6a

                Mtss1

                Tdrd5

                Ccr5

                Gpr55

                Nav1

                Tek

                Ccrl2

                Grap2

                Neb

                Tgfbr2

                Cd14

                H2-DMa

                Nlrp1b

                Tlr1

                Cd180

                H2-DMb2

                Nlrp1c

                Tlr13

                Cd200r2

                H2-Q6,H2-Q8,LOC68395

                Npy1r

                Tlr7

                Cd28

                Hey2

                Nrn1

                Tlr9

                Cd300a

                Hist1h1d

                Oas2

                Tmem106a

                Cd300lb

                Hist1h1e

                Oasl2

                Tmem233

                Cd300ld

                Hist1h2bg

                Olfr1033

                Tmem86a

                Cd86

                Hist2h3b

                Olfr110

                Tnfrsf1b

                Cdh2

                Hist2h4

                Olfr111

                Tns1

                Chst15, Gm10584

                Hist3h2a

                Olfr1392

                Trem1

                Cited4

                Hist4h4

                Olfr1393

                Trim30c

                Clec4a1

                Hivep2

                Olfr915

                Trim30d

                Clec4d

                Hpse

                Olfr916

                Trim58

                Clec4n

                Hsd3b6

                Olfr917

                Trpc6

                Cma1

                Ier2

                Olfr918

                Tsc22d3

                Cma2

                Ifi204

                Orm3

                Tspan13

                Cmklr1

                Ifi27l2a, Ifi27l2b

                P2rx7

                Tspan8

                Creb5

                Ifitm3

                P2ry6

                Tubb2b

                Csf1r

                Ifitm6

                Pcdhga10

                Txk

                Ctnna2

                Ighm

                Pcdhgb6

                Ugt1a10

                Ctsh

                Igk-V28

                Pdzk1ip1

                Unc93b1

                Cx3cr1

                Il18

                Pgap1

                Zbp1

                Cybb

                Il2ra

                Pid1

                Zbtb8a

                Cyp4a12a

                Il6ra

                Pion

                Zfhx3

                Dab2

                Iqsec3

                Pld2

                 

                Darc

                Irf5, Tnpo3

                Pld4

                 

                Dbc1

                Irf8

                Plekhm3

                 
                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 down-regulated 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).
                http://static-content.springer.com/image/art%3A10.1186%2F1471-2407-14-84/MediaObjects/12885_2013_4325_Fig6_HTML.jpg
                Figure 6

                Identification of transcripts dysregulated by miR-9 overexpression in normal murine BMMCs and P815 malignant mast cells. (A) Transcriptional profiling of mBMMCs expressing pre-miR-9-3 lentivirus or empty vector control was performed using Affymetrix GeneChip® Mouse Gene 2.0 ST Arrays to identify genes showing differential expression (>2-fold) with miR-9 overexpression. Real-time PCR was performed to validate changes in gene expression for transcripts (HSPE, TLR7, PERP, PPARG, SLPI) altered by miR-9 overexpression in mBMMCs (*p < 0.05). (B) Transcriptional profiling of P815 mast cells expressing pre-miR-9-3 lentivirus or empty vector control was performed as described above. Real-time PCR was performed to independently validate expression levels of genes (SERPINF1, MLANA, CD200R1, CD200R4) altered by enforced miR-9 expression in P815 cells (*p < 0.05). (C) Mouse BMMCs and P815 cells expressing pre-miR-9-3 lentivirus or empty vector control were collected and real-time PCR for IFITM3, PDZK1IP1, and CMA1 was performed (*p < 0.05). Three independent experiments were performed using cells from 3 separate transduction experiments and all reactions were performed in triplicate.

                Table 5

                Gene transcripts altered by miR-9 overexpression in P815 mast cells

                Upregulated with miR-9 expression (P815)

                Downregulated with miR-9 expression (P815)

                Ifitm3

                Ligp1

                Pdzk1ip1

                Ppm1j

                Cma1

                Gbp2

                Pfkp

                Hist2h3c1

                Serpinf1

                Ly6a

                Trim63

                Cd200r1

                As3mt

                Gzmb

                Speg

                Gbp6

                Mlana

                Afp

                Mgl1

                Ifit1

                Tmem223

                Parp14

                Fjx1

                Ctla2a

                Vamp5

                Igtp

                Cthrc1

                Slamf1

                Ptgis

                Tnfrsf9

                Ass1

                Cpa3

                Ahi1

                Ctla2b

                Akap13

                Tgtp//Tgtp2

                Prf1

                Rabgap1l

                Ston2

                Clec4e

                Hcfc1

                Parp9

                Trak1

                Plekha1

                Ankrd6

                Il1rl1

                Atn1///Rnu7

                Sdf2l1

                Fam122b

                Gvin1

                Mll1

                Il2ra

                Zbtb12

                Fcgr1

                Ahnak

                Gfi1

                Sec14l1

                Thoc1

                Mknk2

                Hist1h2ad

                Apobec2

                Tmed7

                Tspan32

                Ugt1a1

                Hnrnpl

                Taf7l

                Serbp1

                Slc13a2

                Msi2

                Cd200r4

                Myl9

                Vegfc

                Runx2

                Oasl2

                Gstm1

                Socs3

                Epb4.1l4b

                677168///Isg15

                LOC100041694

                Ctso

                2310051F07Rik

                Adam8

                Arx///LOC100044440

                Samd9l

                Mest

                1810014B01Rik

                Mpp4

                LOC641050

                Rp131

                Lrrc28

                Sphk1

                Hist2h2be

                Ebi3

                 

                Igf1

                Bold indicates predicted miR-9 targets.

                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 significantly upregulated CMA1, IFITM3, and PDZK1IP1 transcripts in mouse BMMCs and P815 cells (Figure 6C). These findings provide further support for the notion that miR-9 induces alterations in gene expression that may contribute to the development of an invasive phenotype.

                Discussion

                MiRNAs regulate various biological functions in normal cells such as growth and differentiation, and they are increasingly recognized as playing critical roles in cancer development and progression. Dysregulation of miRNA expression resulting from amplification or loss of miRNAs in tumors compared to their normal tissue counterparts suggests that miRNAs can function as either oncogenes or tumor suppressor genes [13]. Studies evaluating miRNA expression in spontaneously occurring tumors in dogs demonstrate that similar to human cancers, alteration of miRNAs likely contributes to tumorigenesis and that high-throughput methodologies used for the study of miRNAs in human tissues can also be applied to dogs [2632].

                Cutaneous MCTs are the most common skin tumor in dogs; however, little is known regarding mechanisms underlying malignant transformation of these cells. The biological behavior of canine MCTs ranges from relatively benign disease cured with surgical removal to aggressive, highly metastatic tumors ultimately resulting in the death of affected dogs. While the presence of activating KIT mutations helps to explain the behavior of some canine MCTs, little is known regarding the potential role of miRNAs in both normal and malignant mast cells. The purpose of this study was to begin to investigate the potential role of miRNA dysregulation in canine MCTs that exhibit aggressive biologic behavior.

                MiRNA expression profiling of primary canine MCTs identified unique miRNA signatures associated with aggressive MCTs as compared to benign MCTs. The unsupervised hierarchical clustering of primary cutaneous MCTs based on their miRNA expression profiles recapitulated the grouping of the tumors based on their biological behavior, supporting the notion that miRNA dysregulation is associated with the biologic behavior of canine MCTs. Furthermore, we found that miR-9 expression was significantly upregulated in aggressive MCTs compared to benign MCTs. Interestingly, miR-9 was identified as a pro-metastatic miRNA in human breast cancer cell lines through its ability to enhance cell motility and invasiveness in vitro and metastasis formation in vivo[33]. More recently, miR-9 expression was found to be significantly increased in paired primary tumors and distant metastatic sites, suggesting direct involvement of miR-9 in the metastatic process [34, 35]. In concordance with the potential role of miR-9 in malignant mast cell behavior, the BR and C2 canine malignant cell lines expressed high levels of miR-9 compared to normal canine BMMCs. Taken together, these data support the notion that dysregulation of miR-9 may contribute to the aggressive biologic behavior of some canine MCTs.

                While activating KIT mutations clearly contribute to the malignant behavior of mast cells, additional cooperating or initiating genetic defects may be required for the malignant transformation and promotion of the metastatic phenotype [3]. Our data demonstrate that overexpression of miR-9 in the C57 and P815 mouse malignant mast cell lines and normal mouse BMMCs significantly enhanced the invasive behavior of mast cells and indicate that miR-9 induces a pattern of gene dysfunction associated with an invasive phenotype regardless of KIT mutation status.

                While some studies have shown that miR-9 promotes metastasis formation [33, 3639] other contrasting studies suggest that increased expression of miR-9 suppresses metastasis formation [40, 41] and that miR-9 inhibits tumor growth [42]. The opposing roles of miR-9 in various tissues may be explained by the expression of different mRNA targets in distinct cellular and developmental contexts. Indeed, miRNA effects do appear to be cell type/tissue specific and contextual in nature. Previous studies have demonstrated that miR-9 is overexpressed in CDX2-negative primary gastric cancers and miR-9 knockdown inhibits proliferation of human gastric cancer cell lines [43]. In contrast, miR-9 is downregulated in human ovarian tumor cells and overexpression of miR-9 suppresses their proliferation, in part by downregulating NFκB1 [40, 42]. Moreover, miRNA dysregulation may affect only certain aspects of cell behavior. In our studies, miR-9 expression in mast cell lines did not provide a survival advantage or prevent apoptosis, but it did alter the invasive phenotype, supporting the contextual nature of miR-9 induced effects.

                To gain insight into possible mechanisms underlying the observed miR-9-dependent invasive behavior of mast cells, we evaluated the effects of miR-9 expression on the transcriptional profiles of BMMCs and P815 cells. MiR-9 modulated the expression of a large number of gene transcripts, including down-regulation of several putative miR-9 targets identified by computational prediction programs. Furthermore, down-regulation of peroxisome proliferator-activated receptor δ (PPARG) was observed in BMMCs following enforced miR-9 expression, a finding consistent with recent studies demonstrating that regulation of PPARG expression is mediated by miR-9 through direct targeting of its 3’-UTR [25]. To draw firm conclusions regarding direct regulation of target gene expression by miR-9, a functional approach for each gene would be required to validate whether these genes are true miR-9 targets, which although relevant, was outside the scope of this study.

                Overexpression of miR-9 significantly altered gene expression in both BMMCs and P815 cells, however, most gene transcripts affected by miR-9 expression differed between normal and malignant mast cells. These observed differences likely reflect variations in the impact of miR-9 that are dependent on cellular context. In our study, we identified gene transcripts that showed similar changes in expression following miR-9 overexpression in both normal and malignant mast cells and validated several genes demonstrating significant changes in expression (interferon-induced transmembrane protein protein 3, IFITM3; PDZK1 interacting protein 1, PDZK1IP1) or implicated in promoting the metastatic phenotype (mast cell chymase, CMA1). IFITM3 belongs to a family of interferon-induced transmembrane proteins that contribute to diverse biological processes, such as antiviral immunity, germ cell homing and maturation, and bone mineralization. The function of these proteins in mast cells is currently unclear [44]. PDZK1IP1 is a small, non-gycosylated membrane-associated protein that localizes to the plasma membrane and Golgi apparatus. While the function of PDZK1IP1 has not been evaluated in mast cells, overexpression of PDZK1IP1 has been documented in human ovarian, breast, and prostate carcinomas and this strongly correlates with tumor progression [45, 46]. Furthermore, overexpression of PDKZK1IP1 in melanoma cell lines enhances cell proliferation, decreases apoptosis, increases cell migration and is, in part, mediated by an increase in reactive oxygen species (ROS) production [47].

                Chymases are serine proteases possessing chymotrypsin-like activity expressed exclusively by mast cells that promote matrix destruction, tissue remodeling and modulation of immune responses by hydrolyzing chemokines and cytokines [48]. Given the role of chymase in the activation of matrix metalloproteases and extracellular matrix degradation, our findings suggest that miR-9 enhances invasion, in part, through increased expression chymase. Indeed, miR-9 overexpression in normal mast cells resulted in increased expression of CMA1 with a concomitant decrease in the expression of secretory leukocyte peptidase inhibitor (SLPI), a direct inhibitor of chymase [49]. These findings are consistent with the notion that that miR-9 promotes a pattern of gene expression contributing to enhanced invasion and suggests a role for chymase in mediating the biologic functions of miR-9.

                Interestingly, miR-9 modulated the expression of other proteases in normal mast cells, including up-regulation of heparinase (HSPE). Heparinase is an endogylocosidase that functions in the degradation and release of heparan sulfate-bound growth factors [50]. Previous studies have shown that enzymatic cleavage of heparin sulfate by heparinase results in disassembly of the extracellular matrix and basement membrane dissolution, inducing structural modifications that loosen the extracellular matrix barrier and enable cell invasion [51]. Heparinase increases tumor invasion in both cell lines and spontaneous tumor models, through both extracellular matrix remodeling and increased peritumoral lymphangiogenesis [52]. Our data show that normal mast cells overexpressing miR-9 exhibit markedly increased HSPE expression, supporting the assertion that miR-9 may promote the metastatic phenotype by enhancing the proteolytic activity of a number of proteases important in physical remodeling of the extracellular matrix and activate mediators responsible for cell dissemination.

                The present study investigated alterations in gene transcript expression affected by miR-9; however, these changes were not demonstrated at the protein level. Gene expression does not directly correlate with changes at the protein level and miRNAs may suppress protein expression by post-transcriptional silencing mechanisms that are not reflected in transcriptional profiling analyses. Furthermore, inhibition of miR-9 in canine mast cell lines would provide further convincing evidence of its importance in mast cell invasion. As such, identifying proteins altered by miR-9 that promote cell invasion and validating these targets in canine cell lines/tumors represents an area of ongoing investigation.

                Conclusion

                In summary, the work presented here is the first to demonstrate that unique miRNA expression profiles correlate with the biological behavior of canine MCTs. Furthermore, overexpression of miR-9 is associated with aggressive biologic behavior of canine MCTs, possibly through the promotion of a metastatic phenotype as demonstrated by enhanced invasive behavior of normal and malignant mast cells and alteration of gene expression profiles associated with cellular invasion in the presence of enforced miR-9 expression. Future work to dissect the exact mechanisms through which miR-9 exerts the invasive phenotype is ongoing with the ultimate goal of identifying potential druggable targets for therapeutic intervention.

                Declarations

                Acknowledgements

                This study was supported by a grant from the Morris Animal Foundation (D09CA-060), The Ohio State University Targeted Investment in Excellence (TIE) Grant, the National Cancer Institute (P03CA016058), and OSU Center for Clinical and Translational Science (UL1TR000090). Tumor samples were provided by The Ohio State University College of Veterinary Medicine Biospecimen Repository.

                Authors’ Affiliations

                (1)
                Department of Veterinary Clinical Sciences
                (2)
                Department of Veterinary Biosciences
                (3)
                Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University
                (4)
                Division of Hematology and Oncology, Department of Internal Medicine, University of California-Davis

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                53. Pre-publication history

                  1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2407/​14/​84/​prepub

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