Unknown,Transcriptomics,Genomics,Proteomics

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Expression comparison of mutant EGFR-driven gliomas with relapse tumors upon mutant EGFR ablation

ABSTRACT: To determine the roles of oncogenic EGFR signaling in gliomagenesis and tumor maintenance, we generated a novel glioma mouse model driven by inducible expression of a mutant EGFR (EGFR*). Genetic suppression of EGFR* induction led to significant tumor regression and prolonged survival. But in spite of the initial response, the tumors relapsed invariably and propagated independent of EGFR*. We used microarrys to directly compare geen expression of control and relapse tumors and identified gene sets specifically activated in relapse tumors. Control and relasped glioma samples upon mutant EGFR extinction were selected for RNA extraction and hybridization on Affymetrix microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Hongwu Zheng

PROVIDER: E-GEOD-64751 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The goal of this study is to identify the mRNA 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 whole transcriptome analysis, SOLiD fragment colorspace transcriptome reads (50nt) were mapped to the human genome (hg19) and assigned to ensemble transcripts using Bioscope 1.3.1 (Life Technologies). The values of reads per kilobase per million reads (RPKM) were determined by Bioscope 1.3.1 CountTags tool using default parameters. Primary alignments with a minimum mapping quality of 10 and minimum alignment score of 10 were counted. Results: Deep sequencing analysis identified subclasses of mRNAs that were affected by EGFR either under normoxia or hypoxia. EGFR-regulated mRNAs (with Log2 fold-change affected by EGFR M-bM-^IM-% 0.4 or M-bM-^IM-$ -0.4) were sorted and over-lapped with mRNAs that were targeted (based on published data and TargetScan prediction with total context score M-bM-^IM-$ -0.20) by the top miRNA candidates affected by EGFR under hypoxia, resulting in 439 mRNAs that regulated by EGFR and likely targeted by the miRNA candidates in response to hypoxia. Conclusion: Whole transcriptome analysis revealed a novel cluster of mRNAs that are likely regulated by EGFR through miRNAs 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 curstomarized next-generation sequencing, including both small RNA application and whole transcriptome analysis. 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 whole transcriptome RNA profiles.

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.

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Expression comparison of mutant EGFR-driven gliomas with relapse tumors upon mutant EGFR ablation

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