MicroRNAs expression profiling during breast cancer progression
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE38867 | GEO | 2012/06/23
SECONDARY ACCESSION(S): PRJNA169159
REPOSITORIES: GEO
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Project description:MicroRNAs (miRNAs) are known to be deregulated in human breast cancer (BC). The purpose of the current study was to investigate the expression of miRNAs in different stages of BC to assess their biological value in BC progression. MiRNA expression was assessed in a series of BC patients (n=7) with distinct stages of tumour progression (Normal, in-situ (DCIS), primary invasive BC and nodal metastases) to evaluate miRNA differential expression. We used an Agilent miRNA microarray based platform which uses miRBase 16 to screen for 1205 Homo sapiens (hsa) and 144 human viral miRNA candidates. To validate the microarray data, the expression of two deregulated miRNAs was measured by TaqMan quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). This study was conducted Formalin Fixed and Paraffin Embedded (FFPE) specimens from 7 female patients. Twenty M-NM-<m thick FFPE sections were cut and mounted on PALM Membrane Slides. Under direct microscopic visualization, tissues of interest were micro-dissected using PALM non-contact Laser catapulsion instrument (P.A.L.M. Microlaser Technologies, Carl Zeiss Ltd). Total RNA, including miRNAs, was extracted with the TRIzol reagent (Invitrogen, supplied by Fisher Scientific UK Ltd) according to the manufacturerM-bM-^@M-^Ys instructions. Total RNA was quantified with Quant-iT RiboGreen RNA Quantitation Kit (Fisher Scientific UK Ltd), which is an accurate method of measuring total RNA with a detection limit of 0.001-1ng/M-BM-5l. RNA purity and RNA integrity number (RIN) were determined on an Agilent 2100 Bioanalyzer and RNA 6000 NanoLabChip Kits (both Agilent Technologies, Santa Clara, USA), and the RIN of all the samples was >2 but <3. This is expected for FFPE breast samples and is acceptable by Agilent Technologies, Santa Clara, USA, for FFPE samples to undergo miRNA microarray analysis. MiRNAs extracted from laser microdissected tissue components were profiled using Agilent microRNA microarray profiling system (Agilent Technologies, Santa Clara, USA). Total RNA samples were spiked using the MicroRNA Spike-In Kit (Agilent Technologies, Santa Clara, USA) to evaluate efficiencies of labelling and hybridisation. Total RNA was treated with Calf Intestinal Alkaline Phosphatase (CIP), and then 100 ng of total RNA was used per sample to initiate a labelling reaction. Ligation master mix for T4 RNA ligase, which includes Cyanine 3-Cytidine biphosphate (Cyanine 3-pCp) (Complete miRNA Labelling and Hyb Kit, Agilent Technologies, Santa Clara, USA) was used to label the dephosphorylated RNA. Cyanine-3-labelled miRNA samples were hybridised to human miRNA microarrays (Release 16.0, 8x60K) (Agilent Technologies, Santa Clara, USA) at 55M-BM-0C for 20 hrs. Microarray slides were washed with increasing stringency (Gene Expression Wash Buffers, Agilent Technologies, Santa Clara), and subsequently dried with acetonitrile (Sigma-Aldrich, St. Louis, USA). Fluorescent signal intensities were detected on an Agilent Microarray Scanner (Agilent Technologies, Santa Clara, USA) using the Scan Control A.8.4.1 Software (Agilent Technologies, Santa Clara, USA) and extracted from the images with the Feature Extraction 10.7.3.1 Software (Agilent Technologies, Santa Clara, USA).
2012-06-22 | E-GEOD-38867 | biostudies-arrayexpress
Project description:In order to improve our understanding of breast cancer (BC) biology and its prognostication, global miRNA expression profiling was performed using microarray technology on systemically untreated BC patients with long-term follow up. In this study, global miRNA expression profiling using microarray technology was conducted on 56 systemically untreated BC patients with long-term follow up. The arrays used for miRNAs expression profiling comprise a total of 640 probes targeting 328 human miRNAs (hsa-miRs), as well as mouse and rat miRNAs, from the miRBase Sequence Database version 8.0.
2011-07-01 | E-GEOD-28321 | biostudies-arrayexpress
Project description:Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). Over 1,800 miRNA loci are annotated in humans, but it remains largely unknown if and at which sites the pri-miRNAs are cleaved by DROSHA. Here we performed in vitro processing on a full set of human pri-miRNAs (miRBase v21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs based on DROSHA-dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing as well as unproductive cleavage events such as “nick” or “inverse” processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.
2021-07-28 | PXD025767 | Pride
Project description:Estrogen Receptor B (ERB) is a member of the nuclear receptor family of homeostatic regulators that is frequently lost in breast cancer (BC), where its presence correlates with a better prognosis and a less aggressive clinical outcome of the disease. Contrary to ERα, its closest homolog, ERB shows also significant estrogen-independent activities, including the ability to inhibit cell cycle progression and to regulate gene transcription in the absence of the ligand. Investigating the nature and extent of this constitutive activity of ERB in BC MCF-7 cells by miRNA-Seq, we identified 127 miRNAs differentially expressed in ERB+ vs ERB- cells in the absence of ligand, including upregulated oncosuppressor miRs, such miR-30a, and downregulated onco-miRs, like miR-21. In addition, a significant fraction of >1,600 unique proteins identified in these cells by iTRAQ (isobaric Tag for Relative and Absolute Quantitation) quantitative proteomics was either increased or decreased by ERB, revealing regulation of multiple cell pathways by ligand-free receptor. Transcriptome analysis indicates that for a large number of proteins regulated by ERB the corresponding mRNAs are unaffected, including a large number of putative targets of ERB-regulated miRNAs, indicating a central role of miRNAs in mediating BC cell proteome regulation by ERB. Expression of a mimic of miR-30a-5p, a direct target and downstream effector of ERB in BC, led to the identification of several target transcripts of this miRNA, including 11 encoding proteins whose intracellular concentration is significantly affected by unliganded receptor. These results demonstrate a significant effect of ligand-free ERB on BC cell functions via modulation of the cell proteome and suggest that miRNA regulation may represent a key event in the control of the biological and clinical phenotype of hormone-responsive BC by this nuclear receptor.
2014-10-23 | PXD000499 | Pride
Project description:MiRNA profiling from whole blood (EDTA) was used to identify a miRNA profile applicable for early stage breast cancer detection In the study presented here, a consecutively collected, well-defined cohort of 48 patients with early stage breast cancer (invasive ductal carcinoma) at diagnosis was compared with a cohort of 57 healthy individuals for known homo sapiens miRNAs (mirbase 15).
2012-02-02 | E-GEOD-31309 | biostudies-arrayexpress
Project description:Introduction: microRNAs (miRNAs) are short non-coding RNAs that negatively regulate gene expression and may play a causal role in invasive breast cancer. Since many genetic aberrations of invasive disease are detectable in earlier stages, we hypothesized that miRNA expression dysregulation and the predicted changes in gene expression would also be found in early breast neoplasias. Methods: Expression profiling of 365 miRNAs by RT-qPCR was combined with laser-capture microdissection to obtain an epithelial specific miRNA expression signature of normal breast epithelium (n=9) and of paired samples of histologically normal epithelium (HN) and ductal carcinoma in situ (DCIS) (n=16). To determine how miRNAs may control the expression of co-dysregulated mRNAs we also performed gene expression microarray analysis in the same paired HN and DCIS samples and integrated this with miRNA-target prediction. We further validated several target pairs by modulating the expression levels of miRNAs in MCF7 cells and measured the expression of target mRNAs and proteins. Results: Thirty-five miRNAs were aberrantly expressed between RM, HN and DCIS. Twenty-nine miRNAs and 420 mRNAs were aberrantly expressed between HN and DCIS. Combining these two datasets with miRNA-target prediction we identified two established target pairs (miR-195:CCND1 and miR-21:NFIB) and tested several novel miRNA:mRNA target pairs. Over-expression of the putative tumor-suppressor miR-125b, under-expressed in DCIS, repressed the expression of MEMO1, which is required for ErbB2-driven cell motility (also a target of miR-125b); and NRIP1/RIP140, which modulates the transcriptional activity of the estrogen receptor. Knockdown of the putative oncogenic miRNAs miR-182 and miR-183, both highly over-expressed in DCIS, increased the expression of CBX7 (which regulates E-cadherin expression), DOK4, NMT2, and EGR1. Augmentation of CBX7 by knockdown of miR-182 expression, in turn, positively regulated the expression of E-cadherin, a key protein involved in maintaining normal epithelial cell morphology which is commonly lost during neoplastic progression. Conclusions: These data provide the first miRNA expression profile of normal breast epithelium and of pre-invasive breast carcinoma. Further, we demonstrate that altered miRNA expression can modulate gene expression changes that characterize these early cancers. We conclude that miRNA dysregulation likely plays a substantial role in early breast cancer development. Two total samples were analyzed via Affymetrix U133A. Case numbers correspond to individual patients. Each sample is identified by case number, histologic lesion and corresponding microarray ID.
2011-02-17 | E-GEOD-24506 | biostudies-arrayexpress
Project description:This project aims at the detection of specific patterns of miRNAs in peripheral blood samples of MS patients. As controls, blood of donors without known affection have been tested. Using the miRNA patterns we hope to detect a diagnostic pattern for the non-invasive diagnosis of MS. In this study, we compared 20 Multiple Sclerosis samples to 21 control samples using the sanger miRBAse 12.0 miRNA biochip manufactured by febit. Samples were analyzed with the Geniom Realtime Analyzer (GRTA, febit gmbh, Heidelberg, Germany) using the Geniom Biochip miRNA homo sapiens. Each array contains 7 replicates of 866 miRNAs and miRNA star sequences as annotated in the Sanger miRBase 12.0. To benchmark the platform, we tested technical replicates using bought total RNA of brain and liver samples (ambion), achieving a correlation of 0.97.
2010-06-25 | E-GEOD-17846 | biostudies-arrayexpress
Project description:This project aims at the detection of specific patterns of miRNAs in peripheral blood samples of lung cancer patients. As controls, blood of donors without known affection have been tested. Using the miRNA patterns we hope to detect a diagnostic pattern for the non-invasive diagnosis of non-small cell lung carcinoma. In this study, we compared 17 lung cancer samples to 19 control samples using the sanger miRBAse 12.0 miRNA biochip manufactured by febit. Samples were analyzed with the Geniom Realtime Analyzer (GRTA, febit gmbh, Heidelberg, Germany) using the Geniom Biochip miRNA Homo sapiens. Each array contains 7 replicates of 866 miRNAs and miRNA star sequences as annotated in the Sanger miRBase 12.0. To benchmark the platform, we tested technical replicates using bought total RNA of brain and liver samples (ambion), achieving a correlation of 0.97.
2010-06-25 | E-GEOD-17681 | biostudies-arrayexpress
Project description:Introduction: microRNAs (miRNAs) are short non-coding RNAs that negatively regulate gene expression and may play a causal role in invasive breast cancer. Since many genetic aberrations of invasive disease are detectable in earlier stages, we hypothesized that miRNA expression dysregulation and the predicted changes in gene expression would also be found in early breast neoplasias. Methods: Expression profiling of 365 miRNAs by RT-qPCR was combined with laser-capture microdissection to obtain an epithelial specific miRNA expression signature of normal breast epithelium (n=9) and of paired samples of histologically normal epithelium (HN) and ductal carcinoma in situ (DCIS) (n=16). To determine how miRNAs may control the expression of co-dysregulated mRNAs we also performed gene expression microarray analysis in the same paired HN and DCIS samples and integrated this with miRNA-target prediction. We further validated several target pairs by modulating the expression levels of miRNAs in MCF7 cells and measured the expression of target mRNAs and proteins. Results: Thirty-five miRNAs were aberrantly expressed between RM, HN and DCIS. Twenty-nine miRNAs and 420 mRNAs were aberrantly expressed between HN and DCIS. Combining these two datasets with miRNA-target prediction we identified two established target pairs (miR-195:CCND1 and miR-21:NFIB) and tested several novel miRNA:mRNA target pairs. Over-expression of the putative tumor-suppressor miR-125b, under-expressed in DCIS, repressed the expression of MEMO1, which is required for ErbB2-driven cell motility (also a target of miR-125b); and NRIP1/RIP140, which modulates the transcriptional activity of the estrogen receptor. Knockdown of the putative oncogenic miRNAs miR-182 and miR-183, both highly over-expressed in DCIS, increased the expression of CBX7 (which regulates E-cadherin expression), DOK4, NMT2, and EGR1. Augmentation of CBX7 by knockdown of miR-182 expression, in turn, positively regulated the expression of E-cadherin, a key protein involved in maintaining normal epithelial cell morphology which is commonly lost during neoplastic progression. Conclusions: These data provide the first miRNA expression profile of normal breast epithelium and of pre-invasive breast carcinoma. Further, we demonstrate that altered miRNA expression can modulate gene expression changes that characterize these early cancers. We conclude that miRNA dysregulation likely plays a substantial role in early breast cancer development.
2011-02-17 | GSE24508 | GEO
Project description:Introduction: microRNAs (miRNAs) are short non-coding RNAs that negatively regulate gene expression and may play a causal role in invasive breast cancer. Since many genetic aberrations of invasive disease are detectable in earlier stages, we hypothesized that miRNA expression dysregulation and the predicted changes in gene expression would also be found in early breast neoplasias. Methods: Expression profiling of 365 miRNAs by RT-qPCR was combined with laser-capture microdissection to obtain an epithelial specific miRNA expression signature of normal breast epithelium (n=9) and of paired samples of histologically normal epithelium (HN) and ductal carcinoma in situ (DCIS) (n=16). To determine how miRNAs may control the expression of co-dysregulated mRNAs we also performed gene expression microarray analysis in the same paired HN and DCIS samples and integrated this with miRNA-target prediction. We further validated several target pairs by modulating the expression levels of miRNAs in MCF7 cells and measured the expression of target mRNAs and proteins. Results: Thirty-five miRNAs were aberrantly expressed between RM, HN and DCIS. Twenty-nine miRNAs and 420 mRNAs were aberrantly expressed between HN and DCIS. Combining these two datasets with miRNA-target prediction we identified two established target pairs (miR-195:CCND1 and miR-21:NFIB) and tested several novel miRNA:mRNA target pairs. Over-expression of the putative tumor-suppressor miR-125b, under-expressed in DCIS, repressed the expression of MEMO1, which is required for ErbB2-driven cell motility (also a target of miR-125b); and NRIP1/RIP140, which modulates the transcriptional activity of the estrogen receptor. Knockdown of the putative oncogenic miRNAs miR-182 and miR-183, both highly over-expressed in DCIS, increased the expression of CBX7 (which regulates E-cadherin expression), DOK4, NMT2, and EGR1. Augmentation of CBX7 by knockdown of miR-182 expression, in turn, positively regulated the expression of E-cadherin, a key protein involved in maintaining normal epithelial cell morphology which is commonly lost during neoplastic progression. Conclusions: These data provide the first miRNA expression profile of normal breast epithelium and of pre-invasive breast carcinoma. Further, we demonstrate that altered miRNA expression can modulate gene expression changes that characterize these early cancers. We conclude that miRNA dysregulation likely plays a substantial role in early breast cancer development.
2011-02-17 | GSE24506 | GEO