Transcription profiling by array of human MDA-MB-231 breast cancer cell line with or without transfection of putative tumour suppressor gene ITIH5 to study the gene's role in suppressing breast cancer metastatic growth
ABSTRACT: 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 E-MTAB-5081.
Project description:To further investigate the molecular mechanisms by which EVs mediated the abnormal localization of tight junction proteins and adherence junction protein, we performed miRNA microarray analysis of extracellular vesicles isolated from breast cancer cells. miRNA expression in extracellular vesicles was collected from MDA-MB-231-D3H1, MDA-MB-231-D3H2LN, BMD2a and BMD2b breast cancer cell lines.
Project description:To investigate the mRNA expression after extracellular vesicles or miRNA treatement, global gene expression analysis was performed in endothelial cells after the transfection of N.C. or miR-181c and or after the addition of extracellular vesicles from cancer cells. mRNA expression in brain endothelial cells was collected from negative control or miR-181c treatment and or after the addition of extracellular vesicles from MDA-MB-231-D3H1, MDA-MB-231-D3H2LN, BMD2a and BMD2b breast cancer cell lines.
Project description:Acquired drug resistance represents a major challenge in chemo-therapy treatment for various types of cancers. We have found that the retinoid X receptor–selective agonist bexarotene (LGD1069, Targretin) was efficacious in treating chemo-resistant cancer cells. The goal of this microarray study was to understand the mechanism of bexarotene’s role in overcoming acquired drug resistance using human breast cancer cells MDA-MB-231 as a model system and paclitaxel as model compound. After MDA-MB-231 cells were repeatedly treated with paclitaxel for 8 cycles with each cycle including a 3-day treatment with 30 nM paclitaxel and followed by a 7-day exposure to control medium, MDA cells resistant to paclitaxel were developed and their growth was no longer inhibited by paclitaxel treatment. Those MDA cells with acquired drug resistance, when treated with paclitaxel and bexarotene in combination, could regain their sensitivity and their growth were again inhibited. Therefore, RNA samples from parental MDA-MB-231 cells, paclitaxel-resistant MDA cells treated with vehicle, paclitaxel alone or in combination with bexarotene, were used for perform global gene expression profiling with Affymetrix HG-U133A gene chips. Keywords: Drug Treatment MDA-MB-231 cells were exposed to regimens on a 10-day cycle: a 3-day treatment with 30 nM paclitaxel and followed by a 7-day exposure to control medium. Paclitaxel resistant MDA-MB-231 cells (MDA-PR) were established within 8 cycles of such treatment (80 days). These MDA-PR cells were then treated with vehicle control, paclitaxel along, or the combination of 30 nM paclitaxel ( 3 days on and 7 days off) and 1 µM Targretin (10 days on) in a new 10-day cycle for 3 months. Thus, there are four treatment groups, parent MDA cells, MDA-PR, MDA-PR treated with paclitaxel, MDA-PR treated with paclitaxel and bexarotene, and each group had four biological replicates.
Project description:In order to study molecular changes in the stroma from tissue samples it is recommended to separate tumor tissue from stromal tissue. This is particularly relevant to mouse tumor xenograft models where tumor, particularly metastatic tumors, can be small and difficult to separate from the host tissue. In our research we compared qualitatively the ability of high-throughput mRNA sequencing, RNA-Seq, and microarrays to detect tumor (human) and stromal (mouse) expression from mixed tumor-stromal samples in terms of the genes and pathways that are involved in cross-alignment (RNA-Seq) and cross-hybridization (microarrays). Human samples consisted of total RNA obtained from MDA-MB-231 human breast carcinoma cell line and isolated from three independent cultures of sub-confluent MDA-MB-231 cell lines in exponential phase of growth. Mouse samples were obtained from NOD scid gamma mice, and normal lung tissue was harvested from three independent age-matched mice.
Project description:Maintenance of chromatin structure is essential to eukaryotic life; dysregulation is known to be causal for aberrant development and disease. The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. We identified the localization of MBD3, a component of Mi-2/NuRD complex, in two breast cancer cell lines (MCF7 and MDA-MB-231) using ChIP-Seq. MBD3 showed cell-type specific localization with overlap across cell lines being less than 50%. MBD3 localized across gene bodies, peaking around the transcription start site (TSS). Contrary to existing models, MBD3 preferentially associated with CpG rich promoters marked by H3K4me3. These data suggest that MBD3, and by extension the Mi-2/NuRD complex, may have roles in fine tuning expression for active genes. These data represent an important first step in defining regulatory mechanisms by which Mi-2/NuRD complex controls chromatin structure and gene expression. Identification of MBD3 localization in human breast cancer cell lines
Project description:RNA extraction and microarray analysis total RNA from immortalized normal mammary epithelial cells (184A1, MCF-12A), breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, SK-BR-3), BCSC (MDA-MB-231SC, MCF-7SC, XM322, XM607). MDA-MB-231SC and MCF-7SC originating from breast cancer cell lines; XM322 and XM607 derived from clinical specimens which had been described in previous submission (E-MTAB-5057). The miRNA profiling was performed using Agilent miRNA array. Microarray experiments were conducted according to the manufacturer's instructions. To select the differentially expressed genes, we used threshold values of ≥ 2 and ≤ −2-fold change and a Benjamini-Hochberg corrected p value of 0.05. The data was Log2 transformed and median centered by genes using the Adjust Data function of Cluster 3.0 software then further analyzed with hierarchical clustering with average linkage (genes which value more than 100 were evaluated).
Project description:Oct4, a key transcription factor for maintaining the pluripotency and self-renewal of stem cells has been reported previously. It also plays an important role in tumor proliferation and apoptosis, but the role of Oct4 been in tumor metastasis is still not very clear. Here, we found that ectopic expression of Oct4 in breast cancer cells can inhibit their migration and invasion. Detailed examinations revealed that Oct4 up-regulates expression of E-cadherin, indicative of its inhibitory role in epithelial-mesenchymal transition (EMT). RNA-sequence assay showed that Oct4 down-regulates expression of Rnd1. As an atypical Rho protein, Rnd1 can affect cytoskeleton rearrangement and regulate cadherin-based cell-cell adhesion by antagonizing the typical Rho protein, RhoA. Ectopic expression of Rnd1 in MDA-MB-231 cells changes cell morphology which influences cell adhesion and increases migration. It is reported that EMT is accompanied by cytoskeleton remodeling, we hypothesized that Rnd1 may play a role in regulating EMT. Over-expression of Rnd1 can partly rescue the inhibitory effects induced by Oct4, not only migration and invasion, but also in E-cadherin level and cellular morphology. Furthermore, silencing of Rnd1 can up-regulate the expression of E-cadherin in MDA-MB-231 cells. These results present evidence that ectopic expression of Oct4 increases E-cadherin and inhibits metastasis, effects which may be related to Rnd1 associated cell-cell adhesion in breast cancer cells. Examination of mRNA profiles in MDA-MB-231 cells with OCT4 overexpressing
Project description:ERα17p is a synthetic peptide corresponding to the sequence P295LMIKRSKKNSLALSLT311 of the estrogen receptor alpha (ERα) and initially synthesized to mimic its calmodulin binding site. ERα17p was subsequently found to elicit estrogenic responses in E2-deprived ERα-positive breast cancer cells, increasing proliferation and E2-dependent gene transcription. Surprisingly, in E2-supplemented media, ERα17p induced apoptosis and modified the actin network, influencing thereby cell motility. Here, we report that ERα17p 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 manufacturer’s 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:Using microarray, we compared miRNAs expression profiles of MDA-MB-231 cells transfected with myocardin and empty vector (pcDNA3.1) and found that 25 miRNAs were significantly changed in myocardin-transfected groups (17 up-regulated and 9 down-regulated miRNAs). Moreover, we showed that 18 of 25 miRNAs significantly regulated by myocardin were inhibited by ERα in MDA-MB-231 cells. In addition,through a microarray approach, we identify the subset of miRNAs modulated by ERα in MDA-MB-231 cells. Our results determined that ERα may function as tumor-promoter through down-regulating expression of 3 miRNAs (miR-26b, miR-146a and miR-331-3p) in MDA-MB-231 cells. There are 4 samples in this experiment (pcDNA3.1-transfected, myocardin-transfected, ERα-transfected, myocardin plus ERα co-transfecteded groups) and each sample was replicated 3 times. The vector-pcDNA3.1 was used as control.
Project description:We used microarray gene expression analyses to search for epithelial-mesenchymal transition (EMT)-related genes that exhibited the greatest differences in expression in the MZF-1 fragment vector-transfected cells relative to the empty vector-transfected cells. Of the 22,203 genes analyzed in both cell lines, 1209 genes had a two-fold increase and 1557 genes had a two-fold decrease in Hs578T-M(S3) cells (P<0.05), and 1272 genes increased and 1494 genes decreased by a similar amount in MDA-MB-231-M(V4) cells. Combined, 821 of the same genes from both cell lines were up-regulated, and 931 of the same genes from both cell lines were down-regulated. The biological functions of these affected genes were diverse and included 11 EMT-related genes (ITGA5, SERPINE1 GNGI1, SEAP1, TIMP1, FN1, TMEFF1, SNAI2, VIM, CALD1 and MSN) which were down-regulated, and 5 MET-related genes (CDH1, TSPAN13, OCLN, KRT19 and DSP) which were up-regulated. To understand functions of MZF-1/Elk-1 heterodimers, we transfected the binding site-derived peptide to the cells to interrupt heterodimer formation, their DNA binding activity, PKCα expression, cell migration and tumorigenicity were decreased, and the mesenchymal-epithelial transition (MET) was present.