Human primary breast tumors and adjacent non-tumoral tissues were obtained from the Institute of Breast Diseases-FUCAM, Mexico. Biopsies were obtained from breast cancer patient after selection following the regulations approved by the FUCAM Ethics Committee, including patient informed consent for research use. None of the patients recruited in this study received anti-neoplastic therapy prior to surgery. After tumor resection, specimens were embedded in Tissue-Tek and snap-frozen in liquid nitrogen at −80°C until analysis. A pathologist analyzed samples for the amount of tumoral cells and to validate non-tumoral tissues. Tumor tissues containing at least 80% of tumor cells and non-tumoral tissues confirmed by the pathologist were used for subsequent analysis.

Human MDA-MB-231, MDA-MB-453, ZR-75, T47-D and MCF-7 breast cancer cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. Cultures were maintained in a 5% CO₂ humidified atmosphere at 37°C.

Total RNA of tissues and cell lines was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA integrity was assessed using capillary electrophoresis system Agilent 2100 Bioanalyzer with the eukaryotic nano-chip. Only samples with a RIN value of 6 or higher were processed. The expression of miR-18b was performed using microRNA assays (Applied Biosystems, Foster City, CA, USA). Total RNA (100 ng) obtained from clinical specimens and cell lines was reverse transcribed using a looped RT primer specific for miR-18b, 0.15 μl of dNTPs (100 mM), 1.0 μl of reverse transcriptase MultiScribe™ (50 U/μl), 1.5 μl of 10X buffer, 0.19 μl of RNase inhibitor (20 U/μl) and 4.16 μl of RNase-free water. Then, diluted retrotranscription reaction (1:15) was mixed with 10 μl of master mix TaqMan (Universal PCR Master mix, No AmpErase^® UNG, 2X), 7.67 μl of RNase-free water, and 1.0 μl of probe PCR. PCR reaction was done in a GeneAmp System 9700 (Applied Biosystems) as follows: 95°C for 10 min, and 40 cycles at 95°C for 15 sec and 60°C for 1 min. The expression of miR-18b levels were measured by qRT-PCR using the comparative Ct (2^−ΔΔCt) method. RNU44 and RNU48 were used as a control for normalization of data.

MDA-MB-231 and MCF-7 cells were transfected with miR-18b inhibitor (#MH10466; Life Technologies) and scrambled at indicated final concentrations using siPORT™ amine transfection agent (Ambion, Inc., Austin, TX, USA). The miR-18b inhibitor was diluted in 25 μl of Opti-MEM^® to 50–200 nM concentrations, and individually added to wells containing cultured cells in 450 μl of DMEM. Knock-down of miR-18b expression was evaluated by qRT-PCR at 48 h post-transfection.

To analyze the effects of miR-18b inhibition on gene expression we compared the gene expression profiles of MDA-MB-231 cells transfected with miR-18b inhibitor and with siPORT amine transfection agent (control) using DNA microarrays. Total RNA (1 μg) was employed for cDNA synthesis and labeling using Amino Allyl MessageAmp™ II aRNA Amplification kit (Ambion) following manufacturer's instructions. Total RNA obtained 48 h after transfection with empty transfection agent was labeled with Cy3, whereas cDNA from cells transfected with miR-18b inhibitor was Cy5 labeled. Dye incorporation efficiency was analyzed measuring specific absorbance. Samples (500 pmol) were hybridized onto Human Exonic Evidence Based Oligonucleotide (HEEBO) arrays obtained from the Stanford Functional Genomics Facility (Stanford, CA, USA). Each microarray contained 43,000 spots, including 85.8% I.M.A.G.E. consortium clones from the Research Genetics sequence verified clone set and 4.4% control spots, corresponding to 18,141 mapped human genes. Hybridization was performed at 42°C for 18 h. Following hybridization, arrays were washed and scanned using a GenePix 4100A Axon scanner (Axon Instruments, Inc., Union City, CA, USA). Fluorescence ratios were extracted using GenePix 5.0 software (Axon Instruments, Inc.). We defined well-measured spots by: i) having a ratio of normalized signal intensity to background noise of >2 for either the Cy5 signal derived from miR-18b inhibitor transfected cells, or the Cy3 signal derived from empty transfected cells and, ii) with no flag assignation by GenePix software which corresponds to poor spot quality caused by non-reliable hybridization and corruption by noise. We performed an algorithm on Microsoft Excel to accomplish both conditions. Background-subtracted fluorescence log2 ratios were globally normalized for each array, and then mean-centered for each gene. Each microarray experiment was repeated as technical replicates for statistical robustness. Finally, to avoid bias caused by different properties of the two cyanine dyes we balanced the intensity level of both channels using Lowess normalization method included on ArrayNorm software (16).

To evaluate the cell viability after inhibition of miR-18b in MDA-MB-231 and MCF-7 cells a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was performed. Cells (2×10⁴) transfected with miR-18b inhibitor (50 nM), scramble sequence, and siPORT transfection reagent as controls were seeded in 48-well culture dishes and incubated in MTT (1 mg/ml) at 37°C for 4 h. The medium was removed and formazan dye crystals were solubilized with 500 μl isopropanol, 4 mM HCl, NP-40 0.1% for 5 min. Absorbance was measured in a spectrophotometer at 540 nm wavelength. Assays were performed in triplicate.

To evaluate the effect of miR-18b inhibition in MDA-MB-231 and MCF-7 cell migration a scratch/wound-healing assay was performed. Briefly, cells transfected with the miR-18b inhibitor (50 nM), scramble sequence, and siPORT transfection reagent as controls were seeded in triplicate in a 6-well plate and grown to 80% confluence. Twenty-four hours post-transfection a vertical wound was traced in the cell monolayer with a sterile plastic tip. After 4 and 24 h cells were fixed with 4% paraformaldehyde in PBS pH 7.0 and monolayer restoration was imaged through an inverted phase contrast microscope (magnification, ×10). The scratched area was quantified at each time point and the resulting values were graphed. To quantify migratory capacity of cells, transwell chambers (Corning) with 6.5-mm diameter and 8-μm pore size polycarbonate membrane were used. MDA-MB-231 and MCF-7 cells (1×10⁵) transfected with miR-18b inhibitor, scramble or siPORT transfection reagent (mock) were transferred to 0.5 ml serum-free medium and placed in the upper chamber, whereas the lower chamber was loaded with 0.8 ml medium containing 10% FBS. The total number of cells that migrated into the lower chamber was counted after 24 h incubation at 37°C.

Each experiment was performed at least three times, and the results are presented as the mean ± SD. One-way analysis of variance (ANOVA) followed by Tukey's test were used to compare the differences between means. P<0.05 was considered as statistically significant.

In our previous studies, we analyzed a limited set of clinical breast tumors and identified 54 microRNAs that exhibit a significant differential expression between tumor and non-tumor tissues (our unpublished data). Of these, miR-18b was overexpressed in breast cancer and it was selected for further analysis. Here, we studied the miR-18b levels in an independent set of clinical specimens and in five human breast cancer cell lines. We extended our analysis by assaying the miR-18b expression using qRT-PCR TaqMan microRNA assays in clinical specimens obtained from 14 human breast cancer patients who did not receive anti-neoplastic therapy prior to surgery. Tumors were stratified based on clinical subtypes as luminal, HER2-positive and triple negative. Clinical features of breast tumors including hormonal receptors status, tumor size, histology, clinical stage, and tumor grade are summarized in Table I. Our results showed that miR-18b expression was increased in breast tumors in comparison to non-tumoral tissues (Fig. 1A). Particularly, the miR-18b expression average was augmented in luminal (2.5-fold), HER2-positive (4.6-fold), and triple negative (17-fold) tumor subtypes relative to non-tumoral specimens (Fig. 1B). We found no relationship between the expression of miR-18b and clinical characteristics of the patients such as tumor grade, clinical stage, tumor size or histological subtype. Then, we analyzed the miR-18b expression in breast cancer cell lines by qRT-PCR using TaqMan microRNA assays. Results showed that miR-18b levels were significantly increased (2- to 7-fold) in MDA-MB-231, MDA-MB-453, ZR-75 and T47-D cell lines in comparison to non-tumoral breast tissues used as control samples (Fig. 1C). In contrast, miR-18b expression was similar in MCF-7 cell line and normal breast tissues. Interestingly, the triple negative MDA-MB-231 cell line and triple negative breast tumors exhibit the highest miR-18b expression levels. We decided to use the MDA-MB-231 cell line as model for searching potential miR-18b targets.

In order to obtain a comprehensive analysis of cellular transcripts that may represent potential targets of miR-18b in triple negative breast cancer, we studied the effects of its inhibition on the transcriptome of MDA-MB-231 cells. We confirmed the effective knock-down of miR-18b expression by qRT-PCR assays using antagomiRs (data not shown). Total RNA was extracted from cells transfected with miR-18b inhibitor (50 nM) and siPORT amine transfection agent (control) cells, and hybridized to Human Gene Expression 12×135 K DNA microarrays (NimbleGen, Roche). We performed data extraction, normalization of replicates and controls, and selection of spots with a significant modulation (cut-off of ≥1.5-fold) as described in Materials and methods. Our microarray analysis identified 263 genes with a significant modulation (adjusted P-value = 0.05). Of these, 43 genes were downregulated and 220 were upregulated. These results indicated that repression of miR-18b had positive effects in global gene expression of MDA-MB-231 cells, as expected for a negative regulator of gene expression. In Tables II and III we show an overview of the top 20 modulated genes in miR-18b-deficient MDA-MB-231 cells.

Surprisingly, we evidenced the upregulation of 55 OR genes in MDA-MB-231 cells deficient for miR-18b (Table IV). The OR proteins are members of a large family of G-protein-coupled receptors which are expressed not only in the sensory neurons of the olfactory epithelium, but also in various other non-chemosensory tissues (17) and in tumor tissues including prostate cancer where their activation inhibits cell proliferation (18,19). However, the relevance and function of OR genes is still unknown in breast cancer. Here, we found that ten upregulated ORs genes were clustered in 11q12.1 chromosome region and six were grouped in 11q24.2 locus. To understand the coordinated upregulation of these genes, we searched for miR-18b binding sites in the 3′UTR of OR genes using miRanda software (20). Intriguingly, results showed that none of modulated OR genes presented potential miR-18b binding sites. This raises the possibility that OR gene expression could be regulated by a miR-18b-mediated indirect mechanism. To test this hypothesis, we searched for miR-18b binding sites in the 3′UTRs of transcription factors leading to activation of OR genes (21). Interestingly, we found that pancreatic and duodenal homeobox 1 (PDX1), a transcription factor regulating ORs, has potential binding sites for miR-18b. In agreement, we found that PDX1 gene was also up-regulated in our microarray assays (fold change 1.98; P=0.039). These data suggested that OR gene expression could be regulated through a miR-18b-indirect mechanism by targeting transcription factors leading to OR activation. However, further experiments are needed to support these assumptions.

Further analysis of microarray data leads us to the identification of diverse genes involved in cancer whose expression was modulated when miR-18b expression was inhibited in breast cancer cells. In Table V we show an overview of upregulated genes which have been reported previously in different malignancies. These genes have been implicated in tumor growth (CHRM2 and POSTN), cell proliferation (NLRP7 and CHMR2), anoikis (OLFM3), apoptosis (REG1B, SCN3B and POSTN), and angiogenesis (KLK3, CHRM2 and POSTN). Interestingly, eight genes (KIR3DL3, NLRP7, KLK3, OLFM3, SEMG1, CRX, POSTN and CEACAM5) have been shown to be involved in invasion and metastasis of cancer cells. To determine whether the overexpression of these genes was related to direct binding of miR-18b, we searched for potential miR-18b binding sites in their 3′UTR region. Eleven genes were predicted to have binding sites for miR-18b, nine of them with one site and two with more than one binding site. Among these, four genes (KIR3DL3, KLK3, CRX and POSTN) were implicated in invasion and metastasis of cancer cells. For instance, the KLK3 gene encodes prostate-specific antigen (PSA), which is a serin protease that has been established as a tumor marker of prostatic adenocarcinoma. This protein was found elevated in women with renal cell carcinoma (22), it has been shown to exert anti-angiogenic properties, and to present mutations in breast tumors (23). Another upregulated gene, POSTN encodes periostin, a secretory protein which has been suggested to function as a cell adhesion molecule participating in osteoblast recruitment, attachment and spreading (24,25). POSTN was found overexpressed in several cancers with enhanced invasiveness, and correlated with metastasis in colorectal and liver cancers, as well in oral cancer cell lines (26).

Our transcriptional profiling of MDA-MB-231 cells with reduced miR-18b expression revealed that a set of modulated genes have key roles in migration and invasion of cancer cells. In order to evaluate whether miR-18b could be implicated in the regulation of migration properties of breast cancer cells, we performed scratch/wound-healing and Transwell assays in MDA-MB-231 cells with reduced expression of miR-18b. We first evaluated the effect of increasing concentrations of antagomiR-18b in cell viability by MTT assay. No significant changes in cell viability were observed using a range of concentrations (25–200 mM) of miR-18b inhibitor in metastatic MDA-MB-231 cells (Fig. 2A). Similar results were obtained in the MCF-7 breast cancer cell line (Fig. 2E). Then, we performed a scratch/wound-healing assay to evaluate whether the suppression of miR-18b expression could have effects on cancer cell migration. Monolayers of MDA-MB-231 and MCF-7 cells non-transfected and transfected with miR-18b inhibitor were grated, and the wounded areas of the cell monolayer were analyzed after 24 h. Results showed that the formation of monolayers was significantly delayed about 70 and 30% in MDA-MB-231 and MCF-7 cells transfected with miR-18b inhibitor, respectively, in comparison with control cells (Fig. 2B and F). By using Transwell chamber assays, we found that the number of migratory cells that crossed the membrane after 4 and 24 h was reduced in MDA-MB-231 (75%) and MCF-7 (40%) cells in comparison with controls confirming that miR-18b abolishment has negative effects in cell migration (Fig. 2D and H). These findings suggested that miR-18b is able to modulate the expression of genes that are important for migration of cancer cells in vitro.