Investigating the altered brain metastatic transcriptome in breast cancer
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE184869 | GEO | 2021/11/12
REPOSITORIES: GEO
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2023-05-16 | GSE214789 | GEO
Project description:Purpose: The aim of this study is to identify genes that are differentially expressed in the different basal cell populations defined by Lgr5 and Tspan8 (T8) expression from adult mouse mammary glands. Methods: Total RNA was extracted from sorted basal populations defined by Lgr5 and Tspan8 expression from the mammary glands of 10-week-old Lgr5-GFP-IRES-creERT2 female mice. Total RNA (50 ng) for each of the two biological replicates was used to generate libraries for whole-transcriptome analysis following Illumina's TruSeq RNA v2 sample preparation protocol. Libraries were sequenced on an Illumina HiSeq 2000 at the Australian Genome Research Facility (AGRF), Melbourne. At least 20 million 100 bp single-end reads were obtained for each sample. Reads were aligned to the mouse genome mm10 using Rsubread version 1.8.0. The number of reads overlapping each Entrez gene was counted using RefSeq gene annotation and featureCounts. Filtering and normalization used the edgeR package. Genes were filtered as unexpressed if their count per million (CPM) was less than 0.5 in fewer than 2 samples. Compositional differences between libraries were normalized using the trimmed mean of M-values (TMM) method. Differential expression was analyzed using the limma package. Counts were transformed to log2-CPM values with associated precision weights using voom. Differential expression was assessed using moderated t-statistics. Genes were considered to be differentially expressed if they achieved a false discovery rate of 5% and a fold-change in excess of 2.
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Project description:We compare the performance of two library preparation protocols (poly(A) and exome capture) in in vitro degraded RNA samples
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Project description:The aim of this study is to compare human transcriptomes based on NGS (RNA-seq). The transcriptome of neuroblastoma IMR32 was compared with the transcriptome of neuroblastoma IMR32, which was differentiated for 16 days in the presence of 2.5 mkM BrdU. Methods. Human mRNA profiles of 16-day differentiation of IMR32 neuroblastoma and non-differentiated IMR32 neuroblastoma were obtained by deep three-fold sequencing using Illumina NovaSeq. The mapping is read into the human genome (hg38) using the hisat program. On average, about 89-90% of all data received was unambiguously aligned in each library. The htseq-count utility has counted the number of reads that have been matched against known genes (ncbi - entrezID). The obtained values (cpm - countpermillion) for each gene for each library were combined into one matrix for further analysis. Results: Using an optimized data analysis workflow, we matched about 30 million sequence reads per sample to the human genome (hg38). Filtration, normalization by the method (TMM), variance estimation and differentially expressed genes estimation were performed in the edgeR module. Genes in which the cpm did not exceed 1 in any three libraries were considered low expressing.
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Project description:This SuperSeries is composed of the following subset Series: GSE14682: Metastatic breast cancer to the brain: set 1 GSE14683: Metastatic breast cancer to the brain: set 2 Refer to individual Series
2010-06-24 | E-GEOD-14690 | biostudies-arrayexpress
Project description:The objective of this experiment is to identify genes that are regulated by the LRR-receptor-like protein TOO MANY MOUTHS (TMM) to control stomatal precursor (meristemoid) cell fate and behavior. An interesting aspect of the tmm phenotype is that mutant plants overproduce stomata on leaves, but lack stomata entirely on inflorescence stems and in other locations on the plant. Despite the failure to produce mature stomata, mutant stems produce presumptive stomatal precursors through asymmetric divisions. This suggests that normal TMM signaling is required to maintain meristemoid cell fate but not to execute formative asymmetric divisions. Because stem stomata develop in a morphogenetic wave from tip to base, it is possible to harvest tissues that contain meristemoids but not guard mother cells or mature stomata. This provides an opportunity to compare the gene expression profiles of tissues with meristemoids that maintain their cellular identity and ultimately produce stomata (Col gl1), to tissues in which meristemoids form but rapidly lose their cellular identity due to a failure in TMM signaling (tmm-1). Because guard mother cells and stomata are not present in the tissue harvested, genes that change in abundance during these developmental stages will be filtered out. Ultimately we hope to reveal genes that operate downstream of TMM to confer meristemoid cell fate or to provide 'niche factors' suitable for their development into stomata. For analysis, approximately 90 developing inflorescence stem tips were collected and flower buds and meristems removed prior to flash freezing for each replicate sample. The region harvested had been previously determined to contain developing meristemoids but not stomata or guard mother cells by microscopic observation. An identical region was harvested from tmm-1 mutant plants grown at the same time, in the same flat. 4 samples were used in this experiment.
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Project description:Introduction: The prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, calcium-activated potassium (BK) channel is significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene, which encodes α subunit of KCa channels (BK channels) in breast cancer metastasis and invasion. Methods: We performed Global exon array to study the expression of KCNMA1 in metastatic breast cancer in brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of α subunit of KCa channel protein in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether KCa channel inhibition attenuates breast tumor invasion and metastasis using KCNMA1 knockdown with siRNA and biochemical inhibition with IBTX (Iberiotoxin). Results: The Global exon array and RT-PCR showed higher KCNMA1 expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased BK channel activity, leading to greater invasiveness and transendothelial migration, both of which could be attenuated by blocking KCNMA1. Conclusion: Determining the relative abundance of BK channel over expression in breast cancer metastatic to brain and the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy.
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Project description:Brain metastasis is one of the most feared complications of cancer and the most common intracranial malignancy in adults. Its underlying mechanisms remain unknown. From breast cancer patients with metastatic disease we isolated cell populations that aggressively colonize the brain. Transcriptomic analysis of these cells yielded overlapping gene sets whose expression is selectively associated with brain metastasis. The expression of seventeen of these genes in primary breast tumors is associated with brain relapse in breast cancer patients. Some of these genes are also associated with metastasis to lung but not to liver, bone or lymph nodes, providing a molecular basis for the long-observed clinical link between brain and lung metastasis. Among the functionally validated brain metastasis genes, the cyclooxygenase COX-2, the EGFR ligand HB-EGF, and the brain-specific α2-6 sialyltransferase ST6GALNAC5 mediate cancer cell passage through the blood-brain barrier. Other brain metastasis genes encode inflammatory factors and brain-specific proteolytic regulators, suggesting a multifaceted program for breast cancer colonization of the brain. Experiment Overall Design: Two different breast cancer cell lines, MDA-MB-231 and freshly isolated pleural effusion CN34 were used in this study. The MDA-MB-231 group contains three biological replicates of the parental, unselected population, and 4 brain metastatic isolates. CN34 contains 2 biological replicates of the parental, unselected population, and 4 brain metastatic isolates. In each case, the parental population was compared to the brain metastatic isolates to identify gene expression changes associated with the brain metastatic phenotype.
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