Project description:We descrive a joint model of transcriptional activation and mRNA accumulation, using estrogen receptor ERM-NM-1 activation in MCF-7 breast cancer cell line, which can be used for inference of transcription rate, RNA processing delay and degradation rate given data from high-throughput sequencing time course experiments. MCF-7 cells were mock treated or with 10nM 17b-E2 to nine time points (5', 10', 20', 40', 80', 160', 320', 640' and 1280'). Genome-wide identification of RNA polymerase II (RNAPII) occupancy and transcriptome profiling (RNA-seq) following E2 induction of MCF-7 cells Please note that the information in the wig.txt files is in gene-specific coordinates, not chromosomic coordinates, as this is the most sensible format for the associated project/paper.
Project description:We descrive a joint model of transcriptional activation and mRNA accumulation, using estrogen receptor ERα activation in MCF-7 breast cancer cell line, which can be used for inference of transcription rate, RNA processing delay and degradation rate given data from high-throughput sequencing time course experiments.
Project description:Background: The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that is activated by xenobiotic chemicals that function as AHR ligands. The response to xenobiotic AHR ligands is toxicity and the induction of drug metabolizing enzymes. The impact of AHR knockdown on gene expression and pathways in human breast cancer cells in absence of xenobiotic AHR ligands has not been investigated on a genome-wide scale. Methods: MCF-7 cells were used as a model of human breast cancer. The AHR was silenced with short interfering RNA against AHR (siAHR). RNA-sequencing coupled with Ingenuity pathway analysis (IPA) was used to determine the impact of AHR knockdown on gene expression and pathways in the absence of xenobiotic AHR ligands. Western blot analysis and recombinant tumor necrosis factor (TNF) was used to investigate the impact of AHR knockdown on TNF induction of MNSOD expression. Results: We found that the AHR is transcriptionally active in MCF-7 breast cancer cells in the absence of xenobiotic AHR ligands. In total, the expression of 634 genes was significantly changed in AHR knockdown cells compared to controls at a false discovery rate of < 10%. The analysis confirmed that drug metabolizing enzymes were AHR targets; however, we found that AHR also promoted the expression of genes that were not directly related to the metabolism of xenobiotics, such as those involved in lipid and eicosanoid synthesis. Gene pathway analysis of the AHR regulated gene dataset predicted TNF activity to be reduced in AHR knockdown cells. Our finding that AHR knockdown inhibited TNF-stimulated increases in MNSOD expression confirmed this IPA prediction. Conclusions: This is the first gene expression profiling study of AHR knockdown breast cancer cells. Several known and novel AHR targets were identified. The results suggest that endogenous AHR regulation impacts eicosanoid synthesis by regulating gene expression. The IPA prediction of reduced TNF activity in AHR knockdown cells was confirmed by showing that TNF-induced increases in MNSOD expression was inhibited in AHR knockdown cells. The requirement of AHR for MNSOD activation is novel and provides a new link by which AHR may impact reactive oxidative species (ROS) signaling. Expression profiles by mRNA sequencing were generated for human MCF-7 cells transfected with cRNAi (6 replicates) or AHR-siRNA (5 replicates) for 36hr. Sequencing was performed on an Illumina HiSeq 1000 using a 2x100 base paired-end strategy.
Project description:Gene expression profiling of very few or even single cells is of particular interest in many applications. However, detection of a large number of mRNA sequences from a small number of cells is limited by the sensitivity of available methods. High-throughput multiplex reverse transcription followed by PCR amplification (RT-PCR) has much to offer to these studies due to its inherent sensitivity, efficiency and cost-effectiveness. A multiplex RT-PCR based high-throughput gene profiling system is described in this communication. With this system >1000 different mRNA species can be amplified in a single tube to a detectable amount. By using specially designed PCR primers, the long-standing low specificity problem associated with high-throughput gene expression profiling has been solved. Amplified sequences are then resolved by microarray with probes that only hybridize to sequences amplified from mRNA. The method is so sensitive that mRNA in single cells can be reliably detected. Differentially expressed genes identified with the high-throughput approach in the breast cancer cell line, MCF-7, and its drug resistant variant, MCF-7/AdrR, could be validated by a different method. The approach may greatly facilitate the analysis of combinatorial expression of known genes in any cells in many important applications with a limited amount of RNA. Keywords: drug resistence
Project description:We investigated the functions/pathways affected by SPEN knockdown in breast cancer by global expression profiling in a cell model, where the human breast cancer cell line, MCF-7, were transfected with an shRNA targeting SPEN mRNA.
Project description:Transcriptional profiling of human MCF-7 breast cancer cells comparing MCF-7 cells treated with control medium (DMEM/F12 + 0,5% BSA) with MCF-7 cells treated with conditioned medium of cancer-associated adipose tissue (CMCAAT) obtained from 2 breast cancer patients. Goal was to determine the effects of CMCAAT treatment on global MCF-7 gene expression.
Project description:Through the use of antagomiR-122, the knockdown of miR-122 was achieved with an efficiency of 70% in the cell line MCF-7RR with phenotype of acquired resistance to ionizing radiation. To evaluate the transcriptomic landscape resulting from knockdown of miR-122 of radioresistant breast cancer cells MCF-7RR. These assays showed the modulation of genes enrichment in RAS-MAPK and TNFR signaling pathways, inflammatory response and regulation of transcription.
Project description:Homologous recombination-mediated DNA repair deficiency (HRD) predisposes to cancer development, but also provides therapeutic opportunities. Here, we identified an HRD gene signature that robustly predicted HRD status. Unexpectedly, concurrent loss of PTEN in BRCA1-deficient cells might extensively rewire the HR repair network and confer resistance to PARP inhibitor, partially through over-expression of TTK. We used the HRD gene signature as a drug discovery tool and found several PARP-inhibitor-synergizing agents through the connectivity map. Thus gene expression profiling can be used to define the functional status of the HR repair network providing prognostic and therapeutic information. Various shRNAs that target genes involved in homologous recombination (HR) were transfected in MCF-10A non-transformed breast cells lines. Stable HR gene knockdown MCF-10A cells were seeded 200000 at 10 cm plate. Cells were harvested after 48 hours culturing and used for gene expression profiling. The shRNAs that target PTEN or BRCA1 genes were transfected in MCF-10A non-transformed breast cell line by lentiviral particles to generate either single gene knockdown or double gene knockdown. Stable BRCA1, PTEN, and BRCA1_PTEN MCF-10A cells were selected. Scrambled control shRNA-transfected MCF-10A cells were applying as control. All knockdown and control MCF-10A cells were seeded with 2 x 10^5 cells at 10 cm culture plate. Cells were cultured in MCF-10A medium and harvested after 48 hours culturing. mRNA was extracted from collected cells and performing gene expression profiling. Three or four biological replicates were applied. Four biological replicates were applied.
Project description:The study aims to elucidate the effect of histone methyltransferase SMYD3 on gene expression in MCF-7 breast cancer cell line. Knockdown luciferase control v.s. knockdown SMYD3 in MCF-7 breast cancer cell line were conducted. Results identify a large proportion of cell cycle-related genes regulated by SMYD3.
Project description:In order to study transcriptional heterogeneity in response to hormone stimulation in breast cancer, we performed high-throughput single-cell RNA sequencing (scRNA-seq) on 3 breast cancer cell lines that differ in their nuclear receptor expression levels. MCF-7, T-47D and ZR-75-1 cell lines were treated with estrogen and progesterone alone or in combination and collected at different time points. Hash-tagging antibodies were used to identify samples.