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| Status |
Public on Dec 31, 2010 |
| Title |
Identification of the receptor tyrosine kinase AXL in triple negative breast cancer as a novel target for the human miR-34a microRNA (gene expression) |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
Triple negative breast cancer (TNBC) is histologically characterized by the absence of the hormone receptors estrogen and progesterone, in addition to having a negative immunostain for HER-2. The aggressiveness of this disease and lack of targeted therapeutic options for treatment is of high clinical importance. MicroRNAs are short 21- to 23 nucleotide endogenous non-coding RNAs that regulate gene expression by binding to mRNA transcripts, resulting in either decreased protein translation or mRNA degradation. Dysregulated expression of miRNAs is now a hallmark of many human cancers. In order to identify a miRNA/mRNA interaction that is biologically relevant to the triple negative breast cancer genotype/phenotype, we initially conducted a miRNA profiling experiment to detect differentially expressed miRNAs in cell line models representing the triple negative (MDA-MB-231), ER+ (MCF7), and HER-2 overexpressed (SK-BR-3) histotypes. We identified human miR-34a expression as being >3-fold down (from its median expression value across all cell lines) in MDA-MB-231 cells, and identified AXL as a putative mRNA target using multiple miRNA/target prediction algorithms. The miR-34a/AXL interaction was functionally characterized through ectopic overexpression experiments with a miR-34a mimic. In reporter assays, miR-34a binds to the putative target site within the AXL 3’UTR to affect luciferase expression. We also observed degradation of AXL mRNA and decreased AXL protein levels, as well as cell signaling effects on AKT phosphorylation and phenotypic effects on cell migration. Finally, we present an inverse correlative trend in miR-34a and AXL expression for both cell line and patient tumor samples.
Comparison of the changes in gene expression as a result of transfections with miR-34a mimic molecules (representing two different vendors; Qiagen and Dharmacon) in MDA-MB-231 cells.
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| Overall design |
MDA-MB-231 cells were transfected in 6-well dishes (600,000 cells) with either AllStar negative control, Qmimic, or Dmimic at 10 nM final concentration using Lipofectamine™ 2000 (Invitrogen; Carlsbad, CA). All transfections were performed in triplicate. Forty-eight hours post-transfection, total RNA was isolated from each sample using Qiagen’s miRNeasy extraction kit (Qiagen; Germantown, MD). Total RNA samples were sent to the Laboratory of Molecular Technology (National Cancer Institute at Frederick; Frederick, MD), for processing on Affymetrix GeneChip Human Genome U133 Plus 2.0 microarrays (Affymetrix; Santa Clara, CA). Expression values were normalized using Robust Multichip Averaging (RMA). Only gene probes (AllStar vs. Mimic) that passed a log2 1.5-fold change, p < 0.05 threshold using an Empirical Bayes moderated t statistics with a Benjamini-Hochberg correction for the false discovery rate were reported. All analyses were performed with Bioconductor packages AFFYGUI and LIMMA on a R environment.
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| Contributor(s) |
Mackiewicz M, Huppi K, Pitt JJ, Cornelison R, Dorsey T, Ambs S, Caplen NJ |
| Citation(s) |
21814748 |
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| Submission date |
May 14, 2010 |
| Last update date |
Mar 25, 2019 |
| Contact name |
Mark Mackiewicz |
| E-mail(s) |
mackiewiczm@mail.nih.gov
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| Phone |
301-594-6044
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| Fax |
301-480-8878
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| Organization name |
National Institutes of Health
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| Department |
National Cancer Institute
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| Lab |
Genetics Branch
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| Street address |
37 Convent Drive
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platforms (1) |
| GPL570 |
[HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array |
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| Samples (9)
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| GSM543397 |
MB231 transfected with AllStar, biological rep1 |
| GSM543398 |
MB231 transfected with AllStar, biological rep2 |
| GSM543399 |
MB231 transfected with AllStar, biological rep3 |
| GSM543400 |
MB231 transfected with Qmimic, biological rep1 |
| GSM543401 |
MB231 transfected with Qmimic, biological rep2 |
| GSM543402 |
MB231 transfected with Qmimic, biological rep3 |
| GSM543403 |
MB231 transfected with Dmimic, biological rep1 |
| GSM543404 |
MB231 transfected with Dmimic, biological rep2 |
| GSM543405 |
MB231 transfected with Dmimic, biological rep3 |
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| This SubSeries is part of SuperSeries: |
| GSE21834 |
Identification of the receptor tyrosine kinase AXL in triple negative breast cancer as a novel target for the human miR-34a microRNA |
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| Relations |
| BioProject |
PRJNA129329 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE21832_RAW.tar |
42.5 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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