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

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.

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 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.

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).

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).

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.

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.

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.

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.

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.

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).

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.

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 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).

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.