Introduction
Breast cancer is the most common cancer in women worldwide, resulting in 350,000 deaths each year [
1,
2]. Most deaths due to breast cancer are the result of metastasis, demonstrated by the drop in five-year survival from 90% to just 23% in women presenting with metastatic disease [
3]. Metastasis involves epithelial-to-mesenchymal transition (EMT) and cellular changes leading to a more invasive phenotype. These invasive changes are critical steps in breast cancer progression and can lead to treatment failure [
4]. A better understanding of the mechanisms underlying these phenotypic changes will allow improved prediction of those patients susceptible to metastasis as well as improved therapeutic strategies [
1].
Previous studies have suggested a role for microRNAs in regulation of metastasis, invasion, proliferation, cell cycle, growth, differentiation and apoptosis [
5‐
8]. MicroRNAs (miRNAs) are small, non-coding RNA molecules approximately 18 to 25 nucleotides in length [
9]. They comprise approximately 3% of the human genome and regulate approximately 30% of transcripts [
10,
11]. Approximately half of miRNAs have been found in "fragile sites", regions associated with cancer [
10]. miRNAs negatively regulate expression of target genes by binding to the 3'UTR of mRNA transcripts to either cause degradation or prevent translation, depending upon complementarity [
5,
9]. miRNAs can regulate expression of many different types of genes and have been shown to function as both tumor suppressors and oncogenes [
5,
6,
12].
Calin
et al. [
13] were the first to show involvement of aberrant miRNA expression in cancer progression. Since then, many studies have demonstrated that dysregulation of miRNAs have implications in invasion, migration and metastasis in breast cancer [
7,
14,
15]. Our studies have shown that miRNA 510 (miR-510), is elevated in breast tumor samples while absent in the matched non-tumor breast tissue samples [
15]. These studies identify Peroxiredoxin 1 (PRDX1) as a novel direct target of miR-510. PRDX1 is a member of a family of peroxidases with six isoforms known to be involved in protection of cells against oxidative stress [
16,
17]. Deletion of PRDX1 has been shown to promote tumor growth in mice [
18]. It is ubiquitously and highly expressed and functions as a tumor suppressor [
18,
19]. The goal of this study was to investigate the role of miR-510 in breast cancer cell migration and tumor growth and to verify PRDX1 as the direct miR-510 target underlying the mechanism of these phenotypic changes.
Materials and methods
Cell culture and reagents
Human breast cancer cell lines (MCF7, CAMA-1, MDA-MB-231, MCF10A and BT549) were cultured and maintained at 37°C with 5% CO2 in medium supplemented with 10% fetal bovine serum and 100 U of penicillin/streptomycin. MCF7, CAMA-1, MDA MB 231 and HEK293 cells were grown in DMEM media. BT549 cells were grown in RPMI media. MCF10A cells were grown in DMEM:F12 (50:50) media. MCF7 media was supplemented with 1 mM sodium pyruvate, 1 mM sodium bicarbonate, 2 mM L-glutamine, 0.1 mM nonessential amino acids and 0.01 mg/mL insulin. MCF10A media was supplemented with 2 mM L-glutamine, 5% horse serum, 10 μg/mL insulin, 20 ng/mL epidermal growth factor (EGF), 500 ng/mL hydrocortisone, and 10 μg/mL cholera toxin. The breast cancer cell line CAMA-1 was a kind gift of R. Neve (University of California, San Francisco, CA, USA). All other lines were obtained from ATCC (Manassas, VA, USA). Ethical approval for our work with human breast cancer cell lines was not required for our in vitro studies. All tissue culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). shPrdx1 vectors were obtained from the Hollings Cancer Center shRNA core laboratory (Medical University of South Carolina, Charleston, SC, USA).
Immunohistochemistry
Antigen retrieval was done by heating in a microwave oven for 2 × 3 minutes on 30% power in 10 mmol/L citrate (pH 6.0), followed by 30 minutes in a steamer. Sections were washed, treated with 0.3% H2O2 for 30 minutes and non-specific binding was blocked with 2.5% horse serum (ImmPRESS Vector staining kit; Vector Laboratories, Burlington, CA, USA) for 20 minutes and then incubated overnight at 4°C with Ki67 or p-Akt primary antibody at a 1:200 and 1:50 dilution, respectively, in 2.5% normal horse serum in PBS. Overnight incubation at 4°C was followed by 3 × 10-minute washes in PBS, Immpress anti-rabbit secondary antibody was incubated (Vector Laboratories) for 30 minutes at room temperature. After washing with H2O, 3,3'-diaminobenzidine substrate (Sigma, St Louis, MO, USA) was added for two minutes followed by washing in H2O. Slides were counterstained with hematoxylin.
Quantitative reverse transcription PCR
Total RNA from cancer cell lines was extracted using the RNeasyPlus Mini Kit (Qiagen, Valencia, CA, USA). Total RNA measuring 1 μg was reverse transcribed in a 20 μl reaction using iScript (Bio-Rad, Hercules, CA, USA). Real time PCR for gene expression was performed with 5 μl of a 1:20 dilution of reverse transcribed cDNA using the universal probe library (UPL) system (Roche, Nutley, NJ, USA) in a LightCycler 480 (Roche). The cycling conditions were performed as per the manufacturer's instructions. Primer sequences for PRDX1 were: forward 5'-cactgacaaacatggggaagt-3' and reverse 5'-tttgctcttttggacatcagg-3' together with UPL probe #20; and for Akt1 forward 5'- gcagcacgtgtacgagaaga-3' and reverse 5'-ggtgtcagtctccgacgtg-3' together with UPL probe #45. Triplicate reactions were run for each cDNA sample. The relative expression of each gene was quantified on the basis of Ct value measured against an internal standard curve for each specific set of primers using the software provided by the instrument manufacturer (Roche). These data were normalized to GAPDH using the primer sequences: forward 5'-agccacatcgctcagacac-3' and reverse 5'-gcccaatacgaccaaatcc-3' together with UPL probe #60.
Taqman analysis
For microRNA analysis RNA was extracted as described above using the RNeasyPlus Mini Kit from Qiagen. Total RNA measuring 100 ng was reverse transcribed using miR-510 specific primers using the Applied Biosystems (Grand Island, NY, USA) reverse transcription kit as per the manufacturer's instructions. Real time PCR was performed with 1 μl of reverse transcribed cDNA using the TaqMan Assay from Applied Biosystems as per the manufacturer's instructions on the Roche LightCycler 480.
Generation of stable cell lines
The cloning of miR-510 into pSuppressor-neo vector is already described [
15]. For the generation of clonal stable MCF10A cells overexpressing miR-510 (510-1; 510-10; 510-11), pSuppressor-neo vector (Imgenex, San Diego, CA, USA) expressing miR-510 was transfected into MCF10A cells and stable cells were selected in medium containing G418. The wild type 3'UTR of PRDX1 was cloned into the
XbaI site of the pGL3-promoter vector (Promega, Madison, WI, USA) using the primers PRDX1_3UTRf 5'-gcgctctagagcgctgggctgt-3' and PRDX1_3UTRr 5'-gcgctctagagactcatcaaggtctcagt-3'. The sequence complementary to the seed of miR-510 was deleted with the primers PRDX1mutF 5'-ttggtaggaatggcctggcgttgtgggcag-3' and PRDX1mutR 5'-ctgcccacaacgccaggccattcctaccaa-3' using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). All constructs were validated by sequencing at MWG Operon (Huntsville, AL, USA).
Lentiviral stable pools
Stable expression of miR-510 in MCF10A, MCF7 and MDA MB 231 cells was achieved through lentiviral infection. Stable expression was achieved through selection in puromycin (Invitrogen). Lentiviral miR-510 and control vectors (pEZX) were purchased directly from GeneCopoeia (Rockville, MD, USA) and lentiviral preparations were made using the Maine Medical Center Research Institute cell culture and viral vector core (Scarborough, ME, USA).
Oligonucleotide transfection
The miRNA inhibitors (Ambion, Austin, TX, USA) are single-stranded chemically enhanced oligoribonucleotides designed to inhibit the endogenous miRNAs. Cells were transfected with the indicated amounts of oligoribonucleotide using the XtremeGene siRNA reagent as per the manufacturer's instructions (Roche). A total of 48 or 72 h after transfection, cells were harvested for protein or RNA extraction and/or assay.
Plasmid transfection
Transient transfections were performed with the indicated amounts of vector using the XtremeGene HP reagent as per the manufacturer's instructions (Roche). A total of 48 or 72 hours after transfection, cells were harvested for protein or RNA extraction and/or assay.
Luciferase assays
Cells were plated at 50,000 cells per well in a 24-well plate. The pGL3 reporter constructs (0.5 μg, firefly luciferase) were co-transfected with pRL-TK (0.05 μg, Renilla luciferase) using NanoJuice as per the manufacturer's instructions (Novagen, Gibbstown, NJ, USA). Luciferase activity was measured after 48 h using the dual luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well.
Western blot analysis
Cell lysate preparation and Western blot analysis using enhanced chemiluminescence were performed as described previously [
15]. Experimental antibodies include human PRDX1 (Abcam, Cambridge, MA, USA). GAPDH and beta-actin (Abcam) were used as loading controls.
Pacman (haptokinetic) migration track assay
Wells within a two-well chamber slide were pre-coated with 5 μg/mL fibronectin and then overlaid with a field of 1 μm in diameter carboxylate-modified polystyrene fluorescent microspheres (Invitrogen). Cells were then seeded at low density (approximately 4/mm2) in normal growth medium and incubated for a period of 24 h. The ability of the cells to create nonfluorescent tracks was then assessed by fluorescent microscopy and quantified using NIH image. Error bars represent the SD from 10 migration tracks in three separate experiments.
Transwell migration and invasion assay
Cells were seeded into the upper chamber of a Transwell insert pre-coated with 5 μg/ml fibronectin for migration or a BD™Matrigel invasion chamber for invasion, in serum-free medium at a density of 50,000 cells per well (24-well insert; pore size, 8 μM; BD Biosciences, San Jose, CA, USA). Medium containing 10% serum was placed in the lower chamber to act as a chemo-attractant, and cells were further incubated for 4 h (migration) and 24 h (invasion). Non-migratory cells were removed from the upper chamber by scraping with a cotton bud. The cells remaining on the lower surface of the insert were stained using Diff-Quick (Dade Behring, Inc., Newark, DE, USA). Cells were quantified as the number of cells found in five random microscope fields in two independent inserts. Error bars represent the SD from three separate experiments.
Wild-type and miR-510 stably transformed MCF10A cells were seeded at a cell density of approximately 4 cells/mm2 in normal growth media. Cells were incubated as normal, and colonies were counted after 7 to 10 d.
Cell growth assay
Cell growth was measured using the SRB assay [
20]. Cells were plated into each well of a 96- well plate and cells were fixed at the indicated time points with ice-cold 5% trichloroacetic acid (TCA), washed and stained with sulforhodamine B (SRB) and the optical density was measured at 560 nm.
Trypan blue/cell viability assay
Cells were either untreated or treated with 50 μM for 24 hours. Cells were collected by trypsinization and 20 μl was mixed 1:1 with trypan blue and counted on an automated cell counter.
Tumor growth
A total of 1 × 106 MDA-MB-231 cells stably transfected with either miR-510 or scramble control were injected orthotopically into eight-week-old female nude mice. Tumors were measured biweekly with electronic calipers and tumor volume calculated using the formula (L × W2)/2.
In vivo protocol approval
Research protocols were designed and conducted in accordance with the guidelines set by the Institutional Animal Care and Use Committee, Medical University of South Carolina, Approval # ARC-2907.
Statistical analysis
For statistical testing, two-sided paired Student's t-tests were done using an Excel spreadsheet. P-values are given for each individual experiment, but in general, P < 0.05 was considered statistically significant. Error bars represent standard deviations of three independent experiments unless indicated otherwise.
Discussion
Since their discovery, microRNAs have been implicated in many steps of cancer development and progression. They have shown potential roles as predictors of treatment outcomes and microRNA profiling of tumors may have the ability to predict prognosis and identify tumor subtypes [
5]. The Croce group has shown that miRNAs are aberrantly expressed in human breast cancers and that this expression correlated to multiple features of cancer, including estrogen and progesterone receptor status, stage, and indices of proliferation and invasion [
7].
Currently in the literature there are few studies highlighting the role of miR-510. They include its involvement in regulating expression of the serotonin receptor type 3 in enterocytes of colonic mucosa, indicating a role in irritable bowel syndrome [
22,
23], as well as identifying elevated levels of miR-510 in Regulatory T cells (Tregs) from Type 1 diabetic patients [
24]. We have previously published the role of miR-510 in promoting migration, invasion and colony formation in breast cancer cells [
15]. We also observed the levels of miR-510 to be elevated in human breast tumor samples [
15]. This study supports the role of miR-510 functioning as an "oncomir", causing increased migration, invasion and colony formation of non-transformed breast and non-invasive breast cancer cells
in vitro and promoting breast tumor growth
in vivo.
Peroxiredoxin 1 (PRDX1) functions as a tumor suppressor and has a cytoprotective role in breast cells [
18,
25]. PRDX1 contains a miR-510 seed sequence in its 3'UTR and we have validated PRDX1 as a direct target of miR-510 and have shown how regulation of PRDX1 by miR-510 contributes to the migratory phenotype observed in miR-510 over-expressing cells. Cao
et al. showed that loss of PRDX1 promotes PTEN oxidation and activation of Akt [
16]. Multiple targets of miR-510 are predicted to directly target multiple negative regulators and effectors of the Akt signaling pathway and, therefore, a potential mechanism of miR-510-mediated increase in cell proliferation, migration, invasion and tumor growth could be through hyperactivation of the Akt signaling pathway. Indeed, we show
in vitro that overexpression of Akt1 leads to an increase in the expression of miR-510 and that inhibition of Akt1 results in a decrease in the expression levels of miR-510. Furthermore, we show
in vivo that miR-510 expressing tumors have increased activation of the Akt pathway as demonstrated by an increase in Akt phosphorylation, suggesting that a positive feedback loop of this pathway may be occurring in these cells. We have identified a novel role for PRDX1 in the inhibition of migration and demonstrate here that miR-510 mediated negative regulation of Prdx1 is able to inhibit both its role in migration as well as its more well-known role in cellular redox response. However, further investigation of the mechanism of miR-510 mediated negative regulation of PRDX1 is necessary to fully understand their role in tumorigenesis and breast cancer progression.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
QJG, JNM, SGB, LMN and NJM participated in and performed the in vivo and IHC assays. QJG, SGB and LMN performed the luciferase and Western blot assays. QJG, VJF and JNM performed the qPCR assays. NJM performed pathological assessment of tumors. QJG, VJF and DPT performed the functional and rescue experiments. ERC, ACL, DPT and VJF conceived of the study and participated in its design and coordination and, with JNM, drafted the manuscript. All authors read and approved the final manuscript.