Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Multiple platform assessment and isomir analysis of the EGF dependent microRNA-ome by microarray and deep sequencing analysis [small RNA-seq]

ABSTRACT: We have used an integrative high content analysis approach to identify the specific miRNAs implicated in EGF signaling in HeLa cells as potential mediators of cancer mediated functions. We have used microarray and deep-sequencing technologies in order to obtain a global view of the EGF miRNA transcriptome with a robust experimental cross-validation. By applying a procedure based on Rankprod tests, we have delimited a solid set of EGF-regulated miRNAs. After validating regulated miRNAs by RT-qPCR, we have derived protein networks and biological functions from the predicted targets of the regulated miRNAs to gain insight into the potential role of miRNAs in EGF-treated cells. In addition, we have analyzed sequence heterogeneity due to editing relative to the reference sequence (isomirs) among regulated miRNAs. Time course experiment comparing HeLa gene expression in response to EGF analyzed by small RNA-seq using Illumina 36-bp read massively parallel sequencing. Three independent experiments were performed where HeLa cells were serum deprived for 24 hours and were either left untreated or treated with EGF for 6h and harvested for RNA extraction. Thus, a total of 6 samples were analyzed, 3 controls and the 3 respective treated counterparts. These same samples were also analyzed in parallel on two different microarray platforms.

ORGANISM(S): Homo sapiens

SUBMITTER: Lauro Sumoy

PROVIDER: E-GEOD-41359 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor.

Llorens Franc F Hummel Manuela M Pantano Lorena L Pastor Xavier X Vivancos Ana A Castillo Ester E Mattlin Heidi H Ferrer Anna A Ingham Matthew M Noguera Marc M Kofler Robert R Dohm Juliane C JC Pluvinet Raquel R Bayés Mònica M Himmelbauer Heinz H del Rio José Antonio JA Martí Eulàlia E Sumoy Lauro L

BMC genomics 20130601

<h4>Background</h4>Epidermal Growth Factor (EGF) plays an important function in the regulation of cell growth, proliferation, and differentiation by binding to its receptor (EGFR) and providing cancer cells with increased survival responsiveness. Signal transduction carried out by EGF has been extensively studied at both transcriptional and post-transcriptional levels. Little is known about the involvement of microRNAs (miRNAs) in the EGF signaling pathway. miRNAs have emerged as major players i ...[more]

PMID: 23724959

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Project description:Purpose: The goal of this study is to identify the miRNA clusters that are regulated by EGFR under normoxia or hypoxia. Method: Total RNAs were extracted from HeLa cells expressing scrambled control or EGFR shRNA-E1 that cultured under normoxia or hypoxia (1% O2) for 24h. Customized Next-Generation RNA deep sequencing, including both small RNA application and whole transcriptome analysis, was performed according to the standard procedure instructed by Applied Biosystems. For small RNA analysis, library inserts were size selected between 18 and 40nts and analyzed using CLC Genomics Workbench 4.7.1. 35nt colorspace reads were trimmed of adaptor sequence and mapped against human pre-miR sequences (miRBase version 16.0). Values of reads per million mapped reads (RPM) were based on mapped reads with no more than 2 mismatches total. A read was considered to come from a mature miRNA if it mapped to pre-miRNA sequences with no more than three upstream or downstream bases, and missing no more than two upstream or downstream bases from predicted mature or mature* sequences as defined in miRBase version 16.0. All the other pre-miRNA mapped reads were assigned as pre-miRNA signal. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Deep sequencing analysis identified specific miRNA clusters that their maturation (miRNA processing efficacy was reflected by the relative expression of precursor miRNAs affected by EGFR compared with the relative expression of mature miRNAs affected by EGFR) were regulated by EGFR under normixa or specifically in response to hypoxia. Top miRNA candidates that regulated by EGFR in response to hypoxia were further verified by TaqMan and SYBR Green qRT-PCR assays. Conclusion: Next-Generation Deep Sequencing for small RNA analysis revealed a novel function of EGFR involved in miRNA maturation in response to hypoxic stress. RNA profiles of HeLa cells expressing scrambled control (S) or EGFR shRNA-E1 (A1) that cultured under normoxia or hypoxia (1% O2) for 24h were generated by AB SOLiD next-generation sequencing. S: HeLa expressing scrambled control cultured under normoxia; A1: HeLa expressing EGFR shRNA-E1 cultured under normoxia; HS: HeLa expressing scrambled control cultured under hypoxia for 24h; HA1: HeLa expressing EGFR shRNA-E1 cultured under hypoxia for 24h. In total, 4 biological samples with no replicates resulted in 4 small RNA profiles.

Dataset Information

Multiple platform assessment and isomir analysis of the EGF dependent microRNA-ome by microarray and deep sequencing analysis [small RNA-seq]

Publications

Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor.

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