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Correlation between Slug transcription factor and miR-221 in MDA-MB-231 breast cancer cells
© Lambertini et al.; licensee BioMed Central Ltd. 2012
Received: 14 May 2012
Accepted: 28 September 2012
Published: 2 October 2012
Breast cancer and its metastatic progression is mainly directed by epithelial to mesenchymal transition (EMT), a phenomenon supported by specific transcription factors and miRNAs.
In order to investigate a possible correlation between Slug transcription factor and miR-221, we performed Slug gene silencing in MDA-MB-231 breast cancer cells and evaluated the expression of genes involved in supporting the breast cancer phenotype, using qRT-PCR and Western blot analysis. Chromatin immunoprecipitation and wound healing assays were employed to determine a functional link between these two molecules.
We showed that Slug silencing significantly decreased the level of miR-221 and vimentin, reactivated Estrogen Receptor α and increased E-cadherin and TRPS1 expression. We demonstrated that miR-221 is a Slug target gene, and identified a specific region of miR-221 promoter that is transcriptionally active and binds the transcription factor Slug “in vivo”. In addition, we showed that in Slug-silenced cells, wich retained residual miR-221 (about 38%), cell migration was strongly inhibited. Cell migration was inhibited, but to a less degree, following complete knockdown of miR-221 expression by transfection with antagomiR-221.
We report for the first time evidence of a correlation between Slug transcription factor and miR-221 in breast cancer cells. These studies suggest that miR-221 expression is, in part, dependent on Slug in breast cancer cells, and that Slug plays a more important role than miR-221 in cell migration and invasion.
KeywordsSlug miR-221 Epithelial mesenchymal transition Breast cancer
Epithelial cancers such as breast carcinomas and their metastatic progression are mainly directed by a phenomenon referred to as epithelial to mesenchymal transition (EMT) [1, 2]. As well described in several reviews, EMT is supported by the same transcription factors (TFs) including ZEB factors and the Snail family of zinc finger proteins both during embryonic development and the metastatic cascade [1, 3–5]. In addition, specific microRNAs (miRNAs) including miR-206, miR-221/222, miR-200, miR-141, miR-203, miR-130a, have been shown to regulate EMT [6–11].
Mounting evidence indicates that the acquisition of an aggressive cancer phenotype through EMT, as well as other cellular events, may be understood by evaluating the regulatory interplay between TFs and miRNAs [12, 13]. Therefore, recent studies have investigated the interactions among specific miRNAs, TFs and target genes associated with this phenomenon. Direct evidence of these circuits in EMT is still little. Some specific networks have been described including miR-203 – Snai1 , a self-reinforcing loop miR-1/miR-200 via Slug , miR-200/miR-192 – p53 , miR-221/222 – TRPS1 , p53/miR-34 axis , and ZEB/miR-200 .
To investigate the key regulatory networks underlying EMT in breast cancer, we evaluated a potential correlation between Slug (SNAI2) transcription factor and miR-221. The ability of miR-221 and Slug to promote EMT and induce invasiveness in breast cancer cell lines has been documented, but crosstalk between these molecules has not been characterized [3, 17, 20].
Slug is a member of the Snail family of zinc-finger transcription factors, and, together with Snail (SNAI1), acts as a master regulator of EMT. Various studies over the past several years have documented the involvement of Slug in human cancers including leukemias , osteosarcoma , esophageal carcinomas , and breast cancers [3, 24], where Slug expression is strongly correlated with the loss of E-cadherin. Multiple lines of evidence suggest that Slug can be considered a marker of malignancy as well as an attractive target for therapeutic modulation of invasiveness in the treatment of specific cancers [25–28].
miR-221 is often overexpressed in aggressive cancers, increases cell proliferation and protects cancer cells against different apoptotic stimuli [29–31]. Recently, the expression level of miR-221 has been significantly associated with Estrogen Receptor alpha (ERα) status in breast cancer, and several studies have demonstrated that miR-221 directly targets ERα [9, 32, 33]. Breast tumors from patients with high miR-221 plasma levels tend to be ERα-negative, more aggressive and show poorer clinical outcomes than ERα positive cancers . In addition, ERα signaling has been correlated with Slug, and at least two different mechanisms showed that ERα decreases Slug expression [35–37].
In this study, we knocked down Slug and miR-221 in ERα-negative breast cancer cells, MDA-MB-231. We determined a functional correlation between these two molecules demonstrating “in vivo” interaction between Slug and miR-221. Rescue experiments with ectopic expression of miR-221, analysis of the expression of genes involved in breast cancer phenotype, and wound healing assay, suggested that the largest contribution to the invasion ability of the cells and their aggressive phenotype comes from Slug rather than miR-221.
Human breast cancer cell lines MDA-MB-231 and MDA-MB-436 were cultured in Dulbecco’s modified Eagle medium-High Glucose (DMEM-HG) (Euroclone S.p.a., Milan, Italy), supplemented with 10% Fetal Calf Serum (FCS) (Euroclone), 2 mM L-glutamine and 100 U/ml penicillin-streptomycin.
Breast cancer cells were transfected with 30 nM siRNA against Slug (Invitrogen, Carlsbad, CA) , 30 nM antagomiR-221, 50 nM pre-miR-221 precursor (named miR-221 mimic) (Ambion Life Technologies, Grand Island, NY), a non-relevant siRNA (si-Scr) (Medium GC Stealth RNAi Negative Control Duplex, Invitrogen), a non-relevant (miR-Scr) mimic and a non-relevant antagomiR (antagomiR-Scr) (Ambion Life Technologies, Grand Island, NY). For all transfections Lipofectamine RNAiMAX (Invitrogen) was used, following the manufacturer’s instructions. In brief, cells were plated the day before transfections in 12-well plates. Transfected cells were grown up to 6 days in a 37°C incubator with 5% CO2. Total RNA and proteins were extracted, and stored at −80°C for subsequent quantitative RT-PCR or Western Blot measurements. Each treatment used at least triplicate samples.
Total RNA including miRs was extracted from breast cancer cell lines using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction and as previously described . Total RNA was used for reverse-transcription and stored at −80°C. Briefly, cDNA was synthesized from total RNA (500 ng) in a 10 μl reaction volume using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). The reactions were incubated first at 16°C for 30 min and then at 42°C for 30 min followed by inactivation at 85°C for 5 min.
Quantitative real-time PCR for miRNA and mRNA quantification
Quantification of miR-221 and miR-222 was performed using TaqMan MicroRNA Assays (Applied Biosystems), followed by detection with the CFX96TM PCR detection system (Bio-Rad, Hercules, CA). The TaqMan MicroRNA Assay for U6 snRNA (assay ID: 001973; Applied Biosystems) was used to normalize the relative abundance of miR-221 and miR-222. For quantification of Slug, E-cadherin, ERα and TRPS1 mRNAs and pri-miR-221 the appropriate TaqMan probes were purchased from Applied Biosystems using GAPDH reference gene for normalization. Relative expression was calculated using the comparative ΔΔCT method and the change in miRNA or mRNA expression was calculated as fold-change. All reactions were performed in triplicate. The experiment was repeated at least three times.
For western blot analysis, the cells were washed twice with ice-cold PBS and cell lysates were prepared as previously reported . Then, 20 μg of each sample were electrophoresed on a 12% SDS-polyacrylamide gel. The proteins were then transferred onto an Immobilon-P PVDF membrane (Millipore, Billerica, MA). After blocking with PBS-0.05% Tween 20 and 5% dried milk, the membrane was probed with the following antibodies: Slug (L40C6) from Cells Signaling Technology (Danvers, CA, USA), ERα (sc-544), E-cadherin (sc-7870), Vimentin (sc-7558) and p53 (sc-126) from Santa Cruz Biotechnology (Santa Cruz, CA). After washing with PBS-Tween, the membranes were incubated with peroxidase-conjugated anti-rabbit antibody (1:50000) or anti-mouse (1:2000) (Dako, Glostrup, Denmark) in 5% non-fat milk. Immunocomplexes were detected using Supersignal West Femto Substrate (Pierce, Rockford, IL). Anti-IP3K was used to confirm equal protein loading.
Viability analysis (calcein-AM uptake assay)
Viability assay was performed as described previously . For propidium iodide and calcein analysis the cells were visualized under a fluorescence microscope (Nikon, Optiphot-2, Nikon corporation, Japan) using the filter block for fluorescein. Dead cells were stained in red, whereas viable ones appeared in green.
Cell cycle analysis
Cell cycle analysis was performed using fluorescence-activated cell sorting (FACS). Briefly, MDA-MB-231 cells were collected 72 hours after transfection and stained with 25 μg/mL of propidium iodide (Roche Molecular Biochemicals, Indianapolis, IN) in phosphate-buffered saline containing 0.1% bovine serum albumin, 0.05% of Triton X-100, and 50 μg/mL of RNase A. Analysis were carried out using FACS Scan (Becton Dickinson, NJ).
Cell proliferation assay
For growth curves analysis an equal number of cells (approximately 3 x 104) were seeded into 24-well plates. Twenty-four hours after transfection, the cells were harvested and counted by trypan blue exclusion method every day up to three days and at day 6.
Scratch wound assay
Forty-eight-hours after transfection a vertical wound was created in the MDA-MB-231 cell layer using a 20-μL pipette tip. Images were captured at designated times (0 and 24 hours) to assess the rate of gap closure.
Chromatin immunoprecipitation (ChIP) assay
Chromatin immunoprecipitation (ChIP) assays were performed with the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) as previously described . Briefly, MDA-MB-231 cells were grown to 70% confluency in DMEM supplemented with 10% FBS. Cross-linking was performed with 1% formaldehyde at 37°C for 10 min, the cells were washed in ice-cold PBS, and suspended in SDS lysis buffer supplemented with 1× protease inhibitor cocktail (Roche Molecular Biochemicals), for 10 min on ice. Samples were sonicated, diluted 10-fold in dilution buffer, and precleared with 80 μl of salmon sperm DNA-coated protein A-agarose beads; the supernatant was used directly for immunoprecipitation with anti-Slug, (sc-10436), anti-acetyl-H3 (sc-56616) or rabbit Ig λ chain control antibody (sc-33134) (Santa Cruz Biotechnology, INC) overnight at 4°C. Immunocomplexes were mixed with 80 μl of DNA-coated protein A-agarose beads followed by incubation for 1 h at 4°C. Beads were collected and sequentially washed 3 times with 1 ml each of the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8.1, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH-8.1, 500 mM NaCl), LiCl wash buffer (0.25 mM LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris-pH 8.1), and TE buffer. The immunocomplexes were eluted twice by adding a 250 μl aliquot of a freshly prepared solution of 1% SDS, 0.1 M NaHCO3 and the cross-linking reactions were reversed by incubation at 65°C for 4 hrs. Further, the samples were digested with proteinase K (10 mg/ml) at 42°C for 1 hour and DNA was purified in 50 μL of Tris–EDTA with a PCR purification kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. For PCR analysis, aliquots of chromatin before immunoprecipitation were saved (Input). PCR was performed to analyze the presence of DNA precipitated by Slug-specific antibody, and by using specific primers to amplify fragments of the miR-221 and TRPS1 gene promoters. Each PCR reaction was performed with 5 μl of the bound DNA fraction or 2 μl of the Input. The PCR was performed as follows: preincubation at 95°C for 5 min, 30 cycles of 1 min denaturation at 95°C, 1 min annealing at the primers temperature, and 1 min at 72°C, with one final incubation at 72°C for 5 min. No-antibody negative control was included in each experiment.
Data are presented as means ± SEM. For qRT-PCR and cell cycle analysis assays, statistical significance was analyzed by unpaired Student’s t test. p-values ≤ 0.05 were considered statistically significant.
Results and discussion
Correlation between Slug and miR-221 expression
Recruitment of Slug at the miR-221-222 locus
Effect of Slug silencing on specific gene expression
The decrease of ERα and TRPS1 expression is a marker of poor clinical outcome in breast cancers. Therefore, although further investigations are required to better understand the correlation among Slug, miR-221, TRPS1 and ERα, nevertheless, removal of Slug and the consequent down-regulation of miR-221 and reactivation/increase of ERα and TRPS1, may be taken into account for the treatment of ERα-negative breast cancer.
These results suggest that miR-221 down-modulation has not major implications in the phenotype arising from Slug silencing, as ectopic miR-221 expression cannot fully rescue it. In addition, this simultaneous modulation of Slug and miR-221 suggests that silence of Slug could significantly protect cells from progression towards an aggressive phenotype or metastatic stimuli that, in this case, are represented by miR-221 overexpression.
Slug is required for cellular invasion and migration
To better characterize the correlation between Slug and miR-221 at the functional level, the effects of their knockdown on the invasive potential of MDA-MB-231 cells were evaluated using the scratch-wound healing assay that is usually employed to determine in vitro migratory ability of the cells.
Interestingly, complete knockdown of miR-221 expression by transfection with antagomiR-221, significantly attenuated the gap closing in MDA-MB-231 cells, but not as much as that observed in Slug-repressed cells. These findings confirm the role of miR-221 in the cell invasive potential, and its involvement in promoting the EMT phenotype [7, 8], but suggest that the largest contribution to the migratory ability comes from Slug rather than miR-221.
Data from the wound healing assay may in part be explained with the change of cells growth ability, and in part with the change of expression of specific genes. As expected, Slug or miR-221 knocked down cells significantly reduced their proliferation rate compared to control cells (untreated or scrambled cells) (Figure 6B). At the same time, we found that miR-221 knockdown causes a significant but not sufficient decrease of Slug expression (Figure 6C). In fact, residual Slug mRNA (68%) only slightly decreased the level of Slug protein, and consequently E-cadherin expression was almost unaffected, as revealed by Western blot analysis. This molecular evidence supports the higher ability of miR-221-repressed MDA-MB-231 cells to close the wounded area compared to Slug-silenced cells, strengthening our hypothesis that Slug is indeed linked to cancer cell migration and invasion more than miR-221. In addition, as previously reported , we confirm that restoration of ERα could not be achieved by miR-221 knockdown in ERα mRNA-negative cell lines such as MDA-MB-231 (Figure 6C), supporting the notion that ERα is a direct target of miR-221 at the translation level.
Furthermore, data from miR-221 knockdown suggest that unlike Slug, probably one of its negative regulators could be a miR-221 target. While further investigations on a possible Slug / miR-221 circuit are needed, our data suggest that Slug is preferable to miR-221 as potential target to obtain inhibition or slowing down of EMT and metastasis.
Taken together, the results presented here provide for the first time evidence of a correlation between Slug transcription factor and miR-221 in MDA-MB-231 breast cancer cells. However, considering the complexity of EMT phenomenon, further experiments are needed to explore the possible Slug / miR-221 circuit, especially to understand regulatory interactions with potential unknown factors acting as molecular mediators inside the loop. This report suggests that miR-221 is, in part, dependent on Slug in breast cancer cells, and that Slug plays a more important role than miR-221 in cell migration and invasion. Therefore, our evidence may be useful for developing therapeutical approaches for poor prognosis breast cancers.
This work was supported by the Fondazione Cassa di Risparmio di Padova e Rovigo. E.L. is a recipient of a fellowship from the Fondazione Cassa di Risparmio di Ferrara.
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