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Transcription profiling of human breast cancer MDA-MB-231 cells treated with PTHrP and control siRNA

ABSTRACT: Parathyroid hormone-related protein (PTHrP) is overexpressed in many cancer types. This secretory protein can induce hypercalcaemia of malignancy by mimicking the action of calcium-regulating parathyroid hormone (PTH). PTHrP may also be involved in the regulation of migration, invasion, proliferation and apoptosis. It can either stimulate signal cascades, such as the cAMP pathway, or act on not yet defined targets in the nucleus. In this study, we analyzed the effect of PTHrP-specific siRNA on gene expression in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were either transfected with the PTHrP-specific siRNA (siPTHrP) or a control siRNA (siLuc). Differences in gene expression pattern in siPTHrP- vs. siLuc-treated cells were measured by microarray analysis. More than 200 differentially expressed genes were found. Some of these genes have important functions in carcinogenesis. Experiment Overall Design: Three independent transfection experiments were performed. Cells were transfected with siPTHrP or siLuc by electroporation, grown for three days and harvested for isolation of total RNA. RNAs were subjected to microarray analysis. The RNAs of experiment 1 are designated as siLuc 1 and siPTHrP 1, those of experiment 2 as siLuc 2 and siPTHrP 2 etc.. To identify siPTHrP-responsive genes, the siPTHrP- and siLuc-RNAs of the same experiment were compared.

ORGANISM(S): Homo sapiens

SUBMITTER: Dittmer A

PROVIDER: S-ECPF-GEOD-4292 | biostudies-other | 2006 May

REPOSITORIES: biostudies-other

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Project description:We used microarrays to detail the global programme of gene expression for MCF-7 and MDA-MB-231 and revealed the correlation between the methylation state of various genomic components and gene expression level. The expression analyses of the two breast cancer cell lines are a part of the whole study. The summary of our study is as follows: We establish a technique, called modified methylation-specific digital karyotyping (MMSDK) based on methylation-specific digital karyotyping (MSDK) with a novel sequencing approach. Briefly, after a tandem digestion of genomic DNA with a methylation-sensitive mapping enzyme and a fragmenting enzyme, short sequence tags are obtained. These tags are amplified, followed by direct, massively parallel sequencing (Solexa 1G Genome Analyzer). This method allows high-throughput and low-cost genome-wide DNA methylation mapping. We applied this method to investigate global DNA methylation profiles for widely used breast cancer cell lines, MCF-7 and MDA-MB-231, which are representatives for luminal-like and mesenchymal-like cancer types, respectively. By comparison, a highly similar overall DNA methylation pattern was revealed for the two cell lines. However a cohort of individual genomic loci with significantly different DNA methylation profile between two cell lines was identified. Furthermore, we revealed a genome-wide significant correlation between gene expression and the methylation status of gene promoters with CpG islands (CGIs) in the two cancer cell lines, and a correlation of gene expression and the methylation status of promoters without CGIs in MCF-7 cells. Experiment Overall Design: Breast cancer cell lines, MCF-7 and MDA-MB-231, were selected for the study of the impact of DNA methylation on gene expression regulation. Total RNA extraction was performed for both cell lines and hybridization was carried out using Affymetrix microarrays. We developed a modified methylation-specific digital karyotyping (MSDK) to obtain DNA methylation profiling genome wide. Then, we combined the analysis of DNA methylation data and gene expression data to reveal a correlation between epigenetic and transcriptional features genome wide.

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Transcription profiling of human breast cancer MDA-MB-231 cells treated with PTHrP and control siRNA

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