Expression data from WASF3 knockdown stable MDA-MB-231 cells and control cells
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ABSTRACT: We recently showed that inactivation of the WASF3/WAVE3 gene in breast cancer cells results in loss of cell motility and invasion in vitro and metastasis in vivo. To obtain a better understanding of molecular mechanisms of action of WASF3, we have established the stable WASF3 knockdown MDA-MB-231 cells using shRNA strategy. We used microarrays to detail the global programme of gene expression after silencing WASF3 and identified distinct classes of up or down-regulated genes associated with breast cancer cell migration and motility The three stable WASF3 knockdown single clones and three control clones were selected for RNA extraction and hybridization on Affymetrix microarrays. To identify altered gene expression patterns in the knockdown cells, we compared gene expression levels between three different knockdown and three different control clones.
Project description:From our previous data, we found that loss of ATAD3A gene expression in breast cancer cells results in loss of cell motility in vitro and metastasis in vivo. To obtain a better understanding of oncogenic pathway of ATAD3A, we have established the stable ATAD3A knockdown MDA-MB-231 cells using lentiviral strategy. We used the whole genome microarrays to detail the global programme of gene expression after depleting of ATAD3A and identified distinct classes of up or down-regulated metastmir associated with breast cancer cells migration Total RNA was extracted from ATAD3A stable knockdown cells (shATAD3A) and the control cells (shGFP). The labeled RNA was hybridized on U133 plus 2.0 Array. To identify altered gene expression patterns with or without ATAD3A expression, we compared average mRNA expression levels between the ATAD3A knockdown and control MDA-MB-231 cells.
Project description:We examined whether SATB1 functions as a global gene regulator in order to maintain the aggressive phenotype of the MDA-MB-231 cell line. We compared the gene expression profiles between control_shRNA-MDA-MB-231 cells, which express SATB1 at high levels, and SATB1_shRNA1-MDA-MB-231 in which the level of SATB1 was greatly downregulated by RNAi technology. This comparative studies were performed using two different platforms (Codelink and Affymetrix genechip) with two culture conditions either on plastic dish (2D) or on matrigel (3D) which allows cells to form a breast-like morphology only for non-aggressive cells. Keywords: Comparative studies on Control_shRNA and SATB1_shRNA1 expressing MDA-MB-231 from 2D or 3D culture. We examined control_shRNA-MDA-MB-231 cells and SATB1_shRNA1-MDA-MB-231 cells under two culture condition;on plastic dish(2D culture) and on Matrigel coated dish(3D culture). When SATB1 was depleted by RNAi technology, these normally aggressive cells exhibited normal breast like morphology on 3D. We used two different microarray platforms (Codelink and Affymetrix) to make expression data. Initial analysis of data and cross-platform comparison were performed using Codelink expression analysis and GeneSpring software. We provide ratio for control_shRNA/SATB1_shRNA1-MDA-MB-231 cells for 2D and 3D on this series.
Project description:RNA was isolated from ectopically sFRP1-expressing MDA-MB-231 cells and control MDA-MB-231 cells and as well from tumor lysates arising from these cells as nude mouse xenograft. Gene expression profiles for these samples were investigated using Affymetrix arrays. Experiment Overall Design: MDA-MB-231 human breast cancer cells were stably transfected with human sFRP1 encoding vector or empty vector as control. After the selection with antibiotics, three clones of MDA-MB-231/sFRP1 and three clones of MDA-MB-231/control were selected. These six clones were cultured individually in DMEM 10% FCS with 1mg/ml G-418. When cells reached 70-80% confluence, RNA was isolated from the cells. In parallel, the three clones of MDA-MB-231/sFRP1 and the three clones of MDA-MB-231/control were pooled respectively. One million of cells from each pool were suspended in 100ul PBS and injected to fat pads of female balb/c nude mice (6 mice were injected with MDA-MB-231/sFRP1 and 5 mice were injected with MDA-MB-231/control) to do a xenograft experiment. A few - several weeks after, mice were sacrificed when tumor reached a certain size, tumors were taken and RNA was isolated using trizol reagent.
Project description:In order to identify patterns of gene expression that are associated with the putative tumor suppressor gene ITIH5 mediated suppression of breast cancer metastatic growth, we performed a transcriptomic micro-array analysis of the MDA-MB-231 wildtype, three independent highly aggressive MDA-MB-231-mock clones and four independent weak aggressive MDA-MB-231-ITIH5 clones. Related methylation profiling data are found under accession <a href='../E-MTAB-5081/'>E-MTAB-5081</a>.
Project description:Identification of MUC4-associated expression of genes by comparing MUC4 knockdown (MDA-MB-231-shMUC4) and control (MDA-MB-231-SCR). Two-condition experiment, MUC4 knockdown cells vs. control. Biological replicates: 2 control, 2 sh-MUC4 transfected, independently grown and harvested. one replicate per array.
Project description:Background: In previous work we discovered that T lymphocytes play a prominent role in the rise of brain metastases of ER-negative breast cancers. In the present study we explored expressional changes due to T cell contact associated with penetration through the BBB for breast cancer cell lines derived from cancers with various affinities for brain. Methods: Differential expression of proteins was identified by comparing the proteomes of the breast cancer cells before and after co-culture with T cells by using liquid chromatography-mass spectrometry (LC-MS). siRNA was used to silence protein expression in the tumor cells and the artificial BBB model was employed to study the effects on passage of the breast carcinoma cell lines. Results: Mass spectrometry-based proteomics revealed significant alterations in the expression of 35 proteins by the breast cancer cell lines upon T cell contact. Among the proteins is coronin-1A, a protein related to cell motility. Knockdown of CORO1A in the breast cancer cells reduced their ability to cross the artificial BBB to 60%. The effects were significantly less for the cell line derived from breast cancer with affinity for brain. The expression of coronin-1A was confirmed by immunohistochemistry and RT-PCR of 52 breast cancer samples of patients with metastasized breast cancers, with and without brain locations. Lastly, CORO1A upregulation was validated in a publicly available mRNA expression database from 204 primary breast cancers with known metastatic sites. Conclusions: We conclude that T lymphocytes trigger cancer cells to express proteins including coronin-1A thereby facilitating their passage through an in vitro BBB. In addition, a prominent role of coronin-1A in the formation of cerebral metastases in breast cancer patients is strongly suggestive by its upregulation in tissue samples of breast cancer patients with brain metastases.
Project description:Cancer metastasis is a complex mechanism involving multiple processes. In an earlier study, we reported that the levels of serine/threonine phosphatase POPX2 were positively correlated with cancer cell motility through modulating MAPK signaling. Surprisingly, here we found that POPX2 knockdown cells induced more numerous and larger tumor nodules in lungs in longer term animal studies, suggesting that lower levels of POPX2 may favor tumor progression in later stages of metastasis. We hypothesize that POPX2 may do so by modulation of angiogenesis. Secretome analysis of POPX2-knockdown MDA-MB-231 cells using SILAC-mass spectrometry and cytokine array showed that silencing of POPX2 leads to increased secretion of exosomes, which may in turn induce multiple pro-angiogenic cytokines. This study, combined with our earlier findings, suggests that a single ubiquitously expressed phosphatase POPX2 influences cancer metastasis via modulating multiple biological processes including MAPK signaling and exosome-cytokine secretion.
Project description:ERM-NM-117p is a synthetic peptide corresponding to the sequence P295LMIKRSKKNSLALSLT311 of the estrogen receptor alpha (ERM-NM-1) and initially synthesized to mimic its calmodulin binding site. ERM-NM-117p was subsequently found to elicit estrogenic responses in E2-deprived ERM-NM-1-positive breast cancer cells, increasing proliferation and E2-dependent gene transcription. Surprisingly, in E2-supplemented media, ERM-NM-117p induced apoptosis and modified the actin network, influencing thereby cell motility. Here, we report that ERM-NM-117p induces a massive early (3h) transcriptional activity in breast cancer cell line MDA-MB-231. Cells after a 4h incubation with medium containing 10% charcoal stripped FBS were incubated with or without E2 (10-6M) or ERa17p in RPMI 1640 supplemented with 10% charcoal stripped FBS, for 3 hours. Total RNA was isolated using Nucleospin II columns (Macheray-Nagel, Dttren, Germany), according to the manufacturerM-bM-^@M-^Ys instructions. RNA was labeled and hybridized according to the Affymetrix protocol (Affymetrix Gene-Chip Expression Analysis Technical Manual), using the HGU133A plus 2 chip, analyzing a total of 54675 transcripts. Signals were detected by an Affymetrix microarray chip reader.
Project description:Dicer, RNase III endonuclease, is an essential enzyme in miRNA biogenesis that regulates target gene expression, and it has been reported that aberrant expressions of Dicer associate with the clinical outcomes of patients in various cancers. To explore the miRNA differencial expression regulated by Dicer in MDA-MB-231/E1A cells, the microarray profiling analysis was employed to conduct differentially expressed miRNAs in stable MDA-MB-231/vector, MDA-MB-231/E1A, and MDA-MB-231/E1A/shDicer cells. The four groups including vector control, E1A-expressing and Dicer knockdown in E1A-expressing MDA-MB-231 cells were harvested and RNA were isolated. Two independent experiments were performed for each group.