Sample collection and RNA purification
All experiments involving animals were approved by an Institutional Animal Care and Use Committee. Pancreatic β cells were obtained by laser-assisted microdissection (LMD; Leica Microsystems, Milan, Italy) from total pancreas of four groups of RIP-TAg transgenic mice (two animals per time point) at the following stages of β cell tumorigenesis: normal/transgenic β cells (4-weeks of age), hyperplastic islets (6-weeks of age), angiogenic switch/carcinoma in situ islets (8-weeks of age) and invasive β cell carcinoma (10-weeks of age). Total pancreas was surgically isolated, formalin-fixed (10% buffered formalin) and paraffin embedded (FFPE). As controls, non-transgenic mice were sacrificed at the same age and processed as above.
Epithelial cells from matched normal colonic mucosa, dysplastic adenoma, adenocarcinoma or corresponding metastasis were isolated by LMD from FFPE blocks of 16 patients with diagnosis of colorectal carcinoma. Epithelial cells from non-neoplastic, tumor or metastatic tissue samples were similarly isolated by LMD from patients with diagnosis of adenocarcinoma of the breast (n=4), lung (n=4), stomach (n=9), bile ducts (n=2), or parathyroid tumors (n=1). Patients’ characteristics are summarized in . Total RNA was isolated using MasterPure RNA Purification Kit (Epicentre Biotechnologies, Madison, WI, USA) following the manufacturer’s instructions and quantified spectrophotometrically.
Total RNA and TMA blocks were available from a series of 62 cases of hepatocellular carcinoma (HCC). Patients’ associated clinical follow-up data was described previously (Augello et al., 2009
). Informed consent was obtained from all patients, and the study was approved by an Institutional Review Board of the University of Milan, School of Medicine.
RT-PCR-based detection of 132 mature miRs and three mouse-specific reference snoRNAs (SNO 55, 412 and 142) was carried out from β cell samples isolated from RIP-TAg transgenic mice or control, non-transgenic mice using the microRNA Reverse Transcription Kit and gene-specific primers and probes (TaqMan MicroRNA Assays), according to the manufacturer’s specifications. All miRNAs were analyzed in duplicate, and instrument raw data (Ct values) were converted into miRNAs relative quantities (RQs) using the geometrical average of the three snoRNAs as normalization factor. For each miRNA, fold change ratios (FC) were calculated between 6, 8 and 10-week old RIP-TAg transgenic mice and 4-week old RIP-TAg animals. A FC=10 or 0.1 for over- or under-expression, respectively, was assigned as threshold for significant different expression. Significantly different miRs were analyzed in aged-matched non-transgenic mice as described above. The RNU48 was used as a reference snoRNA for human tissues or cell lines. For these experiments, miRNA expression relative to reference was calculated using the 2^(−ΔCt) formula. All reagents, primers, kits and instruments were obtained from Applied Biosystems (Life Technologies, Carlsbad, CA, USA).
k-Ras mutation analysis
Genomic DNA from tumor samples collected from 16 patients with diagnosis of metastatic colon adenocarcinoma was amplified with the following specific primers for k-Ras gene: k-Ras F: 5′-GTACTGGTGGAGTATTT-3′; k-Ras R: 5′-ATACAGCTAATTCAGAATCA-3′. Both strands of k-Ras amplicons were sequenced using BigDye terminators (Applied Biosystems, Life Technologies) and mutations occurring at codons 12 and 13 were identified.
Cells and cell culture conditions
Human colon carcinoma SW1116, hepatocellular carcinoma HepG2 and HUH-7, and breast adenocarcinoma MDA-MB231, MCF-7, SKBR3, and MDA-MB435 cells were purchased from the American Type Culture Collection (ATCC). Human embryonic kidney HEK293 cells were a generous gift from Dr. Locati (University of Milan). Normal human mammary epithelial cells (HMEC) were obtained from Gibco-Invitrogen (Life Technologies). Various cell types were seeded at 2×106 cells/well in 6-wells plates, and transfected with 100 pmol of pre-miR-296 (p-miR-296), antagomir-miR-296 (a-miR-296), pre-miR-1, pre-miR or Cy3-labeled anti-miR negative controls (Ambion, Life Technologies) in the presence of 5 μl Lipofectamine 2000 in 1 ml of OptiMem medium (Gibco-Invitrogen, Life Technologies). Stable miR-296 or control vector (GeneCopoeia, Rockville, MD, USA) expressing MDA-MB231 cells were generated after Lipofectamine 2000 transfection and puromycin (Sigma Aldrich, Milan, Italy) selection for one week. Then cells were trypsinized and plated without selection medium. MDA-MB231 with stable expression of miR-296 were validated by qPCR for recombinant miR expression and used for xenograft studies within passage four of culture.
Xenograft breast cancer model
Twelve female CD1 athymic mice were injected subcutaneously in the fourth mammary fat pad with 2×105 MDA-MB231 cells stably transfected with control miR or miR296 in a total volume of 100 μl of sterile PBS (six mice per condition). Tumor growth was monitored externally using vernier calipers and animals were euthanized after 3 weeks. Tumors were then formalin-fixed and paraffin embedded for histological examination. Tumor volume (mm3) was calculated using the formula: Volume = (width)2 × length/2.
MDA-MB231 cells were transfected with 100pmol of siGENOME or control Smart Pool siRNAs (cat. M-010500, D-001206 all from Dharmacon, Lafayette, CO, USA) and 5 μl of Lipofectamine 2000, as before. Subsequent to transfection, cells were maintained in complete medium for 24 or 48 and processed for individual experiments.
Cell cycle, apoptosis and cell viability assays
Cell cycle transitions and quantification of hypodiploid DNA content (i.e. sub-G1 cell fraction) were determined after 48 or 72 h in transfected cultures by propidium iodide staining and flow cytometry, as described (Romagnoli et al., 2008). Cells transfected with non-targeting constructs were used as unit sample.
Colony formation assay
MDA-MB231 or MCF-7 cells (5×103) were transfected with the various miRNA constructs, and seeded as a single-cell suspension in the presence of 1 ml of 0.7% top agar solution and 1% base agar solid layer after 48 h. Agar for both layers was dissolved in appropriate media supplemented with 10% FBS. Cell were cultured at 37°C, fed twice per week with complete culture medium, and plates were stained after 21 d with 0.5 ml of 0.05% Crystal Violet (Sigma-Aldrich) for 1h. Colonies with a diameter of at least 100 μm were counted by light microscopy and photographed.
Isolation of human breast cancer associated fibroblasts (CAFs)
We isolated tumor-associated fibroblasts from three patients surgically resected for breast cancer. Briefly the tissues were minced with blades and digested as previously described (Bauer et al., 2010
). After isolation, CAFs were plated in cell culture dishes, cultured in 10%-FBS supplemented DMEM media (Gibco-Invitrogen, Life Technologies), and used for invasion assay of MDA-MB231 cells at the second and third splitting passage.
Motility and directional cell migration assays
Subconfluent (70%) cultures of MCF-7, MDA-MB231 or HMEC cells were transfected with the various constructs for 24–48 h. Wounds in the cell monolayer were created using a P200 micropipette tip, after which cells were washed in PBS and incubated in complete medium for 24 h. For migration assay, three random images (50x) were taken at the time of the scratch and after 24 h. The migration distance (units) was determined as reduction in the wound’s gap using NIH Image-J software. For directional cell migration, subconfluent monolayers of MDA-MB231 cells were transfected with the various constructs, scratched as above after 66 h or 42 h, respectively, and analyzed for Golgi orientation after 6 h, by immunofluorescence. The position of the Golgi relative to the nucleus and to the migration front was scored using Image-J software in cells at the leading edge, as described (Dow et al., 2007
; Osmani et al., 2006
; Phua et al., 2009
). Golgi staining was performed using Alexa Fluor555-conjugated antibody to GM130 (BD Bioscience, Milan, Italy). The direction of stress fibers was assessed in the same experiments. F-actin was imaged by phalloidin-TRITC staining (Sigma-Aldrich) using an AxioImager Z1 microscope (Carl Zeiss, Göttingen, Germany) were mounted in DAPI I (Abbott, Abbott Park, IL, USA).
MDA-MB231 or HMEC cells (5×104) transfected with the various constructs were seeded in serum-free medium in Matrigel-coated chambers (8.0 μm pores, BD Biosciences). Cells were allowed to migrate across coated inserts using serum-containing medium or CAFs as chemoattractant for 24 h. The cells on the apical surface of the insert were scraped off, and membranes with invaded cells were fixed in 100% methanol, stained with Toluidine blue (Sigma-Aldrich) and mounted on microscope slides. Invading cells were counted after photographing the membranes, and both the peripheral and the central areas were evaluated.
MCF-7 or MDA-MB231 cells were seeded (2×105) in poly-HEMA (20 mg/ml, Sigma-Aldrich)-coated 6-wells plates, after transfection with the various miRNA-mimics for 48 h or with the indicated siRNA for 24h. Cells were harvested after additional 24 h, and viability was assessed by propidium iodide content and flow cytometry (FACS). When PKH26 staining was performed (Sigma-Aldrich), 2×107 A549 or MDA-MB231 cells were labeled the day before transfection following manufacturer protocol.
miR-296 modulated genes analysis
The mRNA level of candidate genes (n=107, listed in Supplementary Table 1
) was investigated in MDA-MB231 cells transfected with miR-296 or control constructs using TaqMan Gene Expression assays (Applied Biosystems, Life Technologies). TATA-binding protein (TBP) was used as housekeeping gene for target genes relative quantification (RQs) using the 2^(−ΔCt)
formula. RQs were median-normalized and log2 transformed to generate an unsupervised hierarchical clustering. A 1.5 fold cutoff was used to select genes modulated by transfection of anti-miR-296 or pre-miR-296 relative to control, which were further analyzed using Ingenuity Pathway software. Functional profiling using Gene Ontology terms for biological process, molecular function and cellular component were conducted for miR-296 predicted targets identified by at least 4 algorithms (n=145) using GOTM tool (http://bioinfo.vanderbilt.edu/gotm
). As reference gene set we used the human genome, and the top 10 functional categories (most significant p-values obtained using the hypergeometric test adjusted for multiple comparisons) with a minimum number of 4 required genes were identified (Supplementary Table 2
Luciferase miRNA target reporter assay
Mutation in miR-296 seeding sequence within Scrib 3′UTR were created using QuikChangeII XL site directed mutagenesis kit (Stratagene, Agilent Technologies, Santa Clara, CA, USA). Vectors containing full length wild-type or mutated 3′UTR sequences of Scrib gene, or control sequence and dual luciferase reporter genes were purchased from GeneCopoeia. HEK293 cells were co-transfected as described above with 1 μg plasmid containing target gene 3′UTR or control sequence in the presence of 50 nM pre-miR-296, anti-miR-296, control oligonucleotides or unrelated miRNA constructs (anti-let-7g and pre-miR-1). Cells were lysed after 30 h and firefly to Renilla luciferase ratios was measured with a dual luciferase assay (Promega, Milan, Italy) using a Synergy 2 luminometer (BioTek, Winooski, VT, USA). Normalized luciferase ratios were determined for all combinations tested of miRNAs and vectors.
Immunoblotting, Immunohistochemistry and Immunofluorescence
For immunoblotting, aliquots of MDA-MB231, MCF-7, or HMEC cells were harvested 48 or 72 h post-transfection, and solubilized in 150 μl of RIPA buffer supplemented with 1X complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Cell lysates (50 μg) were separated by electrophoresis on 6% or 12% SDS-polyacrylamide gels, transferred to PVDF membranes (Millipore, Billerica, MA, USA), probed with 1 μg/μl of antibodies against Scrib (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-Actin (Sigma-Aldrich), BMP8A (Novus Biologicals Littleton, CO, USA), HMGA1 (LSBio Seattle, WA, USA) or cytokeratin AEI-AE3 (Dako, Milan, Italy), and reactive bands were visualized with ECL Plus reagents (GE Healthcare, Milan, Italy). CAFs were visualized by immunofluorescence staining with an antibody to vimentin (Dako). Immunohistochemical staining of FFPE tissue samples collected from patients with metastatic carcinomas or HCC, was carried out using an antibody to Scrib (10μg/μl, Santa Cruz Biotechnologies) for 1 h at 22°C followed by counterstaining with hematoxylin. As negative control, slides were incubated with dilution buffer instead of primary antibody. Slides were scored by light microscopy and photographed images were arranged with Photoshop for Windows. Cytoplasmatic and membranous Scrib immunoreactivity was scored as negative (0), weak (1), moderate (2) or strong (3) by two authors (GC and SB) independently.
Statistical analysis and clinical validation of miR-296 and Scrib
All experiments were performed in duplicate and performed at least three times. Differences among samples were analyzed using Student’s t-test, unless otherwise specified. The correlation between k-ras mutation status and miR-296 in colorectal carcinoma patients was determined using the Fisher’s exact test. For survival analysis, the Kaplan–Meier method was used. To explore a potential association between miR expression and metastatic relapse, patients were assigned to two groups depending on miR-296 relative expression in tumor versus the averaged non-neoplastic counterparts. A miR-296 ratio of ≤0.5 was used for the “Low” group, whereas a miR-296 value of >0.5 was used for the “High” group. To estimate the association of Scrib mRNA or protein expression to tumor recurrence, HCCs patients were categorized in two groups according to Scrib expression value as for the miR. The two-sided log-rank test was used to compare survival curves. Statistical analyses were performed using GraphPad Prism version 4, or Ministat 2.1 software. A P<0.05 was considered as statistically significant.