MicroRNA-510 promotes cell and tumor growth by targeting peroxiredoxin1 in breast cancer
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MicroRNAs are small non-coding RNAs that are involved in the post-transcriptional negative regulation of mRNAs. MicroRNA 510 (miR-510) was initially shown to have a potential oncogenic role in breast cancer by the observation of its elevated levels in human breast tumor samples when compared to matched non-tumor samples. Few targets have been identified for miR-510. However, as microRNAs function through the negative regulation of their direct targets, the identification of those targets is critical for the understanding of their functional role in breast cancer.
Breast cancer cell lines were transfected with pre-miR-510 or antisense miR-510 and western blotting and quantitative real time PCR were performed. Functional assays performed included cell growth, migration, invasion, colony formation, cytotoxicity and in vivo tumor growth. We performed a PCR assay to identify novel direct targets of miR-510. The study focused on peroxiredoxin 1 (PRDX1) as it was identified through our screen and was bioinformatically predicted to contain a miR-510 seed site in its 3' untranslated region (3'UTR). Luciferase reporter assays and site-directed mutagenesis were performed to confirm PRDX1 as a direct target. The Student's two-sided, paired t-test was used and a P-value less than 0.05 was considered significant.
We show that miR-510 overexpression in non-transformed and breast cancer cells can increase their cell growth, migration, invasion and colony formation in vitro. We also observed increased tumor growth when miR-510 was overexpressed in vivo. We identified PRDX1 through a novel PCR screen and confirmed it as a direct target using luciferase reporter assays. The reintroduction of PRDX1 into breast cancer cell lines without its regulatory 3'UTR confirmed that miR-510 was mediating its migratory phenotype at least in part through the negative regulation of PRDX1. Furthermore, the PI3K/Akt pathway was identified as a positive regulator of miR-510 both in vitro and in vivo.
In this study, we provide evidence to support a role for miR-510 as a novel oncomir. We show that miR-510 directly binds to the 3'UTR of PRDX1 and blocks its protein expression, thereby suppressing migration of human breast cancer cells. Taken together, these data support a pivotal role for miR-510 in breast cancer progression and suggest it as a potential therapeutic target in breast cancer patients.
KeywordsMicroRNA peroxiredoxin1 tumorigenesis breast cancer migration miR-510
3' untranslated region
epidermal growth factor
glyceraldehyde 3-phosphate dehydrogenase
Regulatory T cells
universal probe library.
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 . 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 . 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 .
Previous studies have suggested a role for microRNAs in regulation of metastasis, invasion, proliferation, cell cycle, growth, differentiation and apoptosis [5, 6, 7, 8]. MicroRNAs (miRNAs) are small, non-coding RNA molecules approximately 18 to 25 nucleotides in length . 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 . 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.  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 . 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 . 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).
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.
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 . 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).
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.
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.
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 . 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.
Colony formation assay
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 . 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.
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.
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.
MicroRNA 510 promotes proliferation, migration, invasion and colony formation in vitro
MicroRNA 510 expression is regulated by the AKT Pathway
To determine which signaling pathways were involved in the activation of miR-510 we performed a luciferase reporter screening assay utilizing a construct that contains a functional miR-510 seed sequence site . We treated the cells with a panel of kinase inhibitors and measured luciferase expression compared to untreated controls. We observed an increase in luciferase activity in the cells treated with the PI3K inhibitor LY294002 when compared to the untreated control, suggesting that the PI3K/Akt pathway might be involved in the activation of miR-510 expression (data not shown). To test this hypothesis, we transiently overexpressed Akt1 in MCF10A cells and performed real time PCR to assess miR-510 expression levels (Figure 2D). We observed a significant (approximately seven-fold) increase in miR-510 levels in MCF10A cells overexpressing Akt1 compared to the empty vector control. To examine the role of Akt1 in the regulation of miR-510 expression we transfected MDA MB 175 VII cells, which express miR-510 at relatively high levels, with a short hairpin vector targeting Akt1 (shAkt1; Figure 2E, F). We performed real time PCR to assess miR-510 expression levels (Figure 2F) and observed a decrease in the levels of miR-510 in the shAkt1 transduced cells when compared to the scrambled control. Taken together these data suggest that the PI3K/Akt pathway may function in the activation of miR-510 in breast cancer.
MicroRNA 510 promotes tumor growth in vivo
Peroxiredoxin 1 is a direct target of miR-510
Prdx1 expression inhibits miR-510-mediated cell migration
miR-510 affects the redox function of Prdx1
Prdx1 is an antioxidant protein and, therefore, its primary function within the cell is involved with the regulation of cellular redox response. To assess whether the miR-510 mediated negative regulation of PRDX1 was able to interfere with this primary function, we performed cell viability assay after treatment with H2O2 (Figure 6E, F). MCF10A cells stably infected with miR-510 showed increased sensitivity to treatment with H2O2 when compared to the scrambled control MCF10A cells (Figure 6E). To assess whether miR-510 was able to increase sensitivity to H2O2 to similar levels as when PRDX1 is inhibited we performed cell viability assay with MDA MB 231 cells stably infected with miR-510 or scr control that were transiently transfected with a short hairpin functionally validated to target PRDX1. After 48 h, cells were either untreated or treated with 50 mM H2O2 for 24 h. Cell viability was assessed using the trypan blue exclusion assay. Control cells were resistant to this concentration of H2O2 and showed no cell death. However, transfection with the shPRDX1 led to an increase in sensitivity to H2O2, and we observed a 60 to 70% increase in cell death. Similarly cells overexpressing miR-510 showed an increased sensitivity to H2O2, showing a 30 to 40% increase in cell death. However, no significant increase in sensitivity to H2O2 was observed in cells with shPRDX1 and miR-510 expression when compared to either treatment alone.
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 . 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 .
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 . We have previously published the role of miR-510 in promoting migration, invasion and colony formation in breast cancer cells . We also observed the levels of miR-510 to be elevated in human breast tumor samples . 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 . 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.
MicroRNA 510 is understudied; however, we have strong evidence to support a pivotal role in breast cancer progression. A greater comprehension of the mechanisms involved in miR-510 mediated tumor progression as well as the direct targets mediating these effects are critical to our understanding of its role in cancer. Exploring the role of miR-510 in metastasis may also allow it to be used as a biomarker and predictor of prognosis in patients, providing the next step toward personalized treatment in breast cancer.
We thank Dr Carola Neumann for the PRDX1 and Akt1 constructs, Dr Robin Muise-Helmericks for the shAkt1 construct and Dr Dennis Watson for breast cancer cell lines. This work was supported in part by the shRNA Shared Technology Resource, by pilot research funding from an American Cancer Society Institutional Research Grant awarded to the Hollings Cancer Center, Medical University of South Carolina and by NIH/MSTP Predoctoral Fellowship Training Grant, GM08716.
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