Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL)

ABSTRACT: Cutaneous T-cell lymphomas (CTCL) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult due to a clinical and histological resemblance to benign inflammatory skin diseases. To address if microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we study miRNA expression in 199 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays we show that the most induced- (miR-326, miR-663b, miR-711) and repressed- (miR-203, miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Q-RT-PCR analysis of 104 patients with CTCL and benign skin disorders validates differential expression of 4 out of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A q-RT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity, sensitivity, and a classification accuracy of 95% indicating that miRNAs have a high diagnostic potential in CTCL. miRNA microarray. From each sample 100 ng of total RNA was labeled with Hy3 fluorescent dye using the miRCURY LNA Array power labeling kit (Exiqon, Denmark). All samples were labeled the same day with the same master mix, in order to minimize technical variation. The Hy3-labelled samples were hybridized to miRCURY LNA arrays (v11.0) (Exiqon, Denmark), containing capture probes targeting all human miRNAs registered in the miRBASE version 15.0 at the Sanger Institute. The hybridization was performed overnight at 56Â°C according to manufacturer specifications using a Tecan HS4800 hybridization station (Tecan, Austria). Since it was not possible to hybridize all arrays in one go, samples were randomly split into 5 batches as to minimize day to day variation in the hybridization process. After hybridization the microarray slides were scanned using an Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) at 5Âµm resolution, and the resulting TIFF images were analyzed using the ImaGene 8.0 software on standard settings (BioDiscovery, Inc., USA).

ORGANISM(S): Homo sapiens

SUBMITTER: Rolf SÃ¸kilde

PROVIDER: E-GEOD-31408 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL).

Ralfkiaer Ulrik U Hagedorn Peter H PH Bangsgaard Nannie N Løvendorf Marianne B MB Ahler Charlotte B CB Svensson Lars L Kopp Katharina L KL Vennegaard Marie T MT Lauenborg Britt B Zibert John R JR Krejsgaard Thorbjørn T Bonefeld Charlotte M CM Søkilde Rolf R Gjerdrum Lise M LM Labuda Tord T Mathiesen Anne-Merete AM Grønbæk Kirsten K Wasik Mariusz A MA Sokolowska-Wojdylo Malgorzata M Queille-Roussel Catherine C Gniadecki Robert R Ralfkiaer Elisabeth E Geisler Carsten C Litman Thomas T Woetmann Anders A Glue Christian C Røpke Mads A MA Skov Lone L Odum Niels N

Blood 20110824 22

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show ...[more]

PMID: 21865341

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Project description:Increasing evidences support a potential role for the Signal Transducer and Activator of Transcription-3 (STAT3) as a tumor driver in cutaneous T-cell lymphoma (CTCL). However, the mechanisms leading to STAT3 pathway activation in CTCL and how STAT3 activation contributes to lymphomagenesis remains primarily not enough explored. Recently, we found that miR-124, a known STAT3 regulator in cancer, is robustly silenced in Mycosis Fungoides (MF) tumor-stage and CTCL cell lines and we have herein studied whether deregulation of miR-124 contributes to activate STAT3 pathway in CTCL. Material and Methods: DNA methylation status of miR-124 and its expression levels in response to the DNA-demethylating agent azacitidine were evaluated in CTCL cell lines by pyrosequencing analysis. CTCL cell lines were transient transfected with a lentiviral vector encoding miR-124 and transfected cells were analyzed for phosphorylated STAT3 (P-STAT3) levels. The impact on STAT3 signaling was evaluated using expression microarray on CTCL cell lines in both conditions, miR-124 absent (malignant condition) versus miR-124 expressed (lentiviral transduction). Results: A significant promoter methylation and silencing of miR-124 in MF tumor samples and CTCL cell lines was detected. DNA methylation levels of miR-124 in CTCL cell lines were restored (and subsequently miR-124 were overexpressed) after DNA demethylation. Ectopic lentiviral expression of miR-124 downregulated STAT3 in the different CTCL cell lines. Genes differentially expressed regarding miR-124 regulation in CTCL cell lines indicated impact on known cell survival and function pathways and, particularly, intervention on miR-124 expression modulated different STAT3 pathway components. Conclusions: Our study deciphers a novel epigenetic mechanisms regulating STAT3 pathway in CTCL. This might contribute to a better understanding the molecular basis in CTCL development. Deregulation of STAT3 seems to have a major impact on cell survival in CTCL cell lines indicating the potential interest of targeting STAT3 with specific therapies.

Project description:Background: Exosomes are nanovesicles of endocytic origin believed to be involved in communication between cells. Recently, it has been shown that mast cell exosomes contain RNA named "exosomal shuttle RNA". The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34 positive progenitor cells. Results: Exosomes from the human mast cell line HMC-1 contain RNA. The exosomes contain no or very little ribosomal RNA compared to their donor cells. The mRNA and microRNA content in exosomes and their donor cells was examined using microarray analyses. We found 116 microRNA in the exosomes and 134 microRNA in the cells, from which some were expressed at different level. DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. Transfer experiments revealed that exosomes and their RNA can transfer to other HMC-1 cells and to CD34 positive progenitors. Conclusions: To conclude, HMC-1 exosomes contain mRNA and microRNA that can be transferred to other mast cells and to CD34 progenitors. This shuttle of exosomal RNA may represent a powerful mode of communication between cells where cells send genetic information to other cells over a distance via exosomes. [miRNA profiling] Identification of microRNA was performed by Exiqon (www.exiqon.com). Briefly, the quality of the total RNA was verified by an Agilent 2100 Bioanalyzer. Total RNA from the exosome and the HMC-1 cell samples were labelled with Hy3 and Hy5 fluorescent stain, respectively, using the miRCURY Hy3/Hy5 power labelling kit. The Hy3-labelled exosome samples and a Hy5-labeled mast cells were mixed pair-wise and hybridized to the miRCURY? LNA array (v9.2). The hybridization was performed according to the miRCURY? LNA array manual using a Tecan HS4800 hybridization station (Tecan Systems, Inc. San Jose, CA). The miRCURY? LNA array microarray slides were scanned by a ScanArray 4000 XL scanner (Packard Biochip Technologies, Billerica, MA ,USA) and the image analysis was carried out using the ImaGene 6.1.0 software (BioDiscovery, Inc, El Segundo, CA USA). The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. MicroRNA with signals equal to or below the background signal in 2 or more of the 4 replicate measurements were identified as absent in that slide. The limit for a miRNA to be listed as detectable was set to signal intensities higher than 3 x background (3 x median Hy3 or Hy5 for the total slide). In addition, where signals were detected for <3 of the slides, they were considered unreliable and excluded from sets of detected miRNAs. The experiment was performed in triplicate samples. The signal was calculated as the mean value of the log2MeanRatio Hy3/Hy5 for the triplicates ± SD.

Dataset Information

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL)

Publications

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL).

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