Human MCF7 cells: Mock control vs. siRNA transfected against calreticulin
Ontology highlight
ABSTRACT: Transcriptional profiling of human MCF7 cells comparing mock control MCF7cells with MCF7cells transfected by siRNA against calreticulin. The latter was shown to be of having significantly decreased expression of calreticulin followed by significant decrease in migrative and invasive potentials of the MCF7 cells. Goal was to determine the effect of calreticulin knockdown on the global gene expression of MCF7 cells. Two-condition experiment, Mock control MCF7 cells vs. calreticulin siRNA knockdown MCF7 cells. Biological replicates: 3 mock control replicates, 3 transfected replicates.
Project description:We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha. However, the relevance of estrogen receptor-beta in mediating endoxifen action has yet to be explored. Therefore, the goals of this study were to determine the differences in the global gene expression profiles elicited by estradiol treatment and endoxifen between parental MCF7 breast cancer cells (expressing estrogen receptor alpha only) and MCF7 cells stably expressing estrogen receptor beta. Total RNA was isolated from parental or estrogen-receptor beta expressing MCF7 cells following 24 hour treatments with either ethanol vehicle, 1nM 17-beta-estradiol or 1nM estradiol plus 40nM endoxifen. All studies were conducted in biological replicates of 2.
Project description:We subjected MCF7 cells to starvation with 0.5% charcoal treated serum for 48h and then we added 17-beta estradiol (E2) at final concentration of 10 nM, profiling before and after 60 minutes of treatment the transcriptome and the translatome, coming from the polysomal pool of mRNAs after sucrose gradient separation. Comparison of translatome profile changes with corresponding transcriptome profile changes represents a way of studying translational control networks and the degree of concordance between cellular controls affecting mRNA abundance and cellular controls affecting mRNA availability to translation. It is well known that E2 is a strong transcriptional regulator, while its translational control activity is less characterized. To provide a direct experimental evaluation of E2 induced translational regulation, we compared translatome and transcriptome profiles of E2 treated cells. Keywords: polysomal profiling, translatome profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, estradiol stimulation, estrogen receptor. The comparison between transcriptional and polysomal profiling was used for the discovery of general and mRNA-specific changes in the translation state of the serum starved MCF7 cells transcriptome in response to E2 stimulus. To identify translationally regulated mRNA molecules, gene expression signals derived from the polysomal populations were compared by microarrays analysis to those obtained from unfractionated total RNAs. Polysomal RNA and total RNA were isolated from MCF7 cells serum starved and treated with E2. Cells lysates were collected before (t = 0 min) and after (t = 60 min) E2 treatment. All experiments were run in quadruplicates.
Project description:We have previously demonstrated that endoxifen is the most important tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing estrogen receptor-alpha. The goals of this study were to compare the gene expression profiles elicited by endoxifen to that of other anti-estrogens in MCF7 cells. We also examined the gene expression profiles elicited by various endoxifen concentrations in the presence of tamoxifen and its other primary metabolites in order to better understand the molecular contributions of endoxifen to the effects of tamoxifen. Total RNA was isolated from parental MCF7 cells following 24 hour treatments with various individual or combined ligands. All studies were conducted in replicates of 3.
Project description:Affymetrix microarray data was generated from MCF7 breast cancer cells treated in vitro with siRNAs against 78 transcription factors and signalling molecules. Gene expresion of 78 functionally important molecules were knocked down in MCF7 breast cancer cells using siRNA. Then the gene expression profiles of these MCF7 cells, along with untreated cells and non-targetting control (SICONTROL) treated cells were analysed using microarrays.
Project description:Full title: Expression data from antisense miRNA-221/222 (si221/222) and control inhibitor (GFP) treated fulvestrant-resistant breast cancer cells The expression of miR-221/222 were found to be upregulated in fulvestrant resistant breast cancer cells MCF7-FR compared to its drug-sensitive counterpart MCF7. To investigate the role of miR-221/222 in acquired resistance to fulvestrant, we lowered the level of miR-221/222 in MCF7-FR cells using miRNA inhibitors (antagomirs), and compared gene expression profiles before and after treatment. Fulvestrant-resistant breast cancer cells MCF7-FR (originated from drug-sentitive breast cancer model cell line MCF7) were transient-transfected by antigomirs targeting miR221 or miR222 (i.e. si221, si222). All three cell lines, MCF7-FR, siR221, siRNA222 were subjected to gene expression profiling.
Project description:Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a Total RNA (tot) was extracted from MCF7 vector cells after 16h of treatment with Doxorubicin (1.5uM) and Nutlin-3a (10uM) or DMSO (solvent, as control treatment). Polysomal profiling was performed after the same conditions. We collected all subpolysomal mRNA fractions (sub) and the polysomal ones (pol) after sucrose gradient fractionation of cytoplasmic lysates to analyze separately mRNAs that are not actively translated from those that are considered in active translation, respectively. Experiments were performed in three biological replicates.
Project description:ABL1 kinase inhibitors such as imatinib mesylate (IM) are effective in managing chronic myelogenous leukemia (CML) but incapable of eliminating leukemia stem cells (LSCs), suggesting that kinaseM-bM-^HM-^Rindependent pathways support LSC survival. Given that the bone marrow hypoxic microenvironment supports hematopoietic stem cells, we investigated if hypoxia similarly contributes to LSC persistence. Importantly, we found that while BCRM-bM-^HM-^RABL1 kinase remained effectively inhibited by IM under hypoxia, apoptosis became partially suppressed. Furthermore, hypoxia enhanced the clonogenicity of CML cells, as well as their efficiency in repopulating immunodeficient mice, both in the presence and absence of IM. HIF1M-bM-^HM-^RM-NM-1, which is the master regulator of the hypoxia transcriptional response is expressed in the bone marrow specimens of CML individuals. In vitro, HIF1M-bM-^HM-^RM-NM-1 is stabilized during hypoxia and its expression and transcriptional activity can be partially attenuated by concurrent IM treatment. Expression analysis demonstrates at the whole transcriptome level that hypoxia and IM regulate distinct subsets of genes. Functionally, knockdown of HIF1M-bM-^HM-^RM-NM-1 abolished the enhanced clonogenicity during hypoxia. Taken together, our results suggest that in the hypoxic microenvironment, HIF1M-bM-^HM-^RM-NM-1 signaling supports LSC persistence independently of BCRM-bM-^HM-^RABL1 kinase activity. Thus targeting HIF1M-bM-^HM-^RM-NM-1 and its pathway components may be therapeutically important for the complete eradication of LSCs. 24 samples consisting CD34+ bone marrow aspirates of 3 chronic phase patients that were subjected to 24h or 96h of DMSO/Normoxia (21% oxygen, 5% carbon dioxide) control, 2 M-BM-5M Imatinib, hypoxia (0.5% oxygen, 5% carbon dioxide) or combined Imatinib/hypoxia treatments in triplicate cultures.
Project description:MCF7 cells were stimulated with vehicle or 100nM corticotropin releasing hormone (CRH) for 24h. The effect of CRH on the expression of genes relevant to estrogen signalling was investigated by using the Human Estrogen Receptor Signaling RTM-BM-2 ProfilerM-bM-^DM-" PCR Array (SABioscience Corp). qPCR gene expression profiling. MCF7 cells were treated separately in triplicate. Equal amount total RNA was processed further for gene expression analysis.
Project description:Transcriptional profiling of Caco-2 cells co-cultured with Faecalibacterium prausnitzii DSM17677, Lactobacilus rhamnosus HN001, UV-killed F. prausnitzii, or no bacteria in an apical anaerobic environment for four hours. 2 colour microarray, reference design. Biological replicates: 6 per treatment group.
Project description:Transcriptional profiling of human MCF7 cells comparing mock control MCF7cells with MCF7cells transfected by siRNA against calreticulin. The latter was shown to be of having significantly decreased expression of calreticulin followed by significant decrease in migrative and invasive potentials of the MCF7 cells. Goal was to determine the effect of calreticulin knockdown on the global gene expression of MCF7 cells.