Comment[ArrayExpressAccession] E-GEOD-47610 MAGE-TAB Version 1.1 Public Release Date 2013-06-04 Investigation Title Development of microRNA expression signatures in laryngeal tumors Comment[Submitted Name] Development of microRNA expression signatures in laryngeal tumors Experiment Description Background: MicroRNA (miRNA) is an emerging subclass of small non-coding RNAs that regulates gene expression and has a pivotal role for many physiological processes including cancer development. Recent reports revealed the role of miRNAs as ideal biomarkers and therapeutic targets due to their tissue- or disease-specific nature. Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, and laryngeal cancer has the highest incidence in it. However, the molecular mechanisms involved in laryngeal cancer development remain to be known and highly sensitive biomarkers and novel promising therapy is necessary. Methodology/Principal Findings: To explore laryngeal cancer-specific miRNAs, RNA from 5 laryngeal surgical specimens including cancer and non-cancer tissues were hybridized to microarray carrying 723 human miRNAs. The resultant differentially expressed miRNAs were further tested by using quantitative real time PCR (qRT-PCR) on 43 laryngeal tissue samples including cancers, noncancerous counterparts, benign diseases and precancerous dysplasias. Significant expressional differences between matched pairs were reproduced in miR-133b, miR-455-5p, and miR-196a, among which miR-196a being the most promising cancer biomarker as validated by qRT-PCR analyses on additional 84 tissue samples. Deep sequencing analysis revealed both quantitative and qualitative deviation of miR-196a isomiR expression in laryngeal cancer. In situ hybridization confirmed laryngeal cancer-specific expression of miR-196a in both cancer and cancer stroma cells. Finally, inhibition of miR-196a counteracted cancer cell proliferation in both laryngeal cancer-derived cells and mouse xenograft model. Conclusions/Significance: Our study provided the possibilities that miR-196a might be very useful in diagnosing and treating laryngeal cancer. To explore laryngeal cancer-specific miRNAs, RNA from 5 laryngeal surgical specimens including cancer and non-cancer tissues were hybridized to microarray carrying 723 human miRNAs. Total RNA including low molecular weight RNA from tissue samples was isolated using the mirVanaTM miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. The quality of the RNA samples was assessed using an Agilent 2100 Bioanalyzer, and only the samples meeting the criteria of 28S/18S > 1 and RNA Integrity Number (RIN) M-bM-^IM-% 7.5 were used for all analyses. For microarray analysis, we used the Human miRNA Microarray Kit V2 (Agilent Technologies, Santa Clara, CA), which contains 20-40 features targeting each of 723 human miRNAs (Agilent design ID 019118) as annotated in the Sanger miRBase, release 10.1. Labeling and hybridization of total RNA samples were performed according to the manufacturer's protocol. One hundred ng of total RNA was used as an input into the labeling reaction, and the entire reaction was hybridized to each array for 20 hours at 55M-BM-0C. The results were analyzed using Agilent GeneSpring GX7.3. Normal controls and cancer samples were compared using Welch's t-test (p<0.05) and differentially expressed miRNAs with at least a 2-fold change in expression were considered to be potential biomarkers. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Minoura Saito Person First Name Kaho Koichiro Person Email geo@ncbi.nlm.nih.gov Person Affiliation Agilent Technologies Japan Ltd Person Address Agilent Technologies Japan Ltd, 9-1,Takakura-cho, Hachioji, Tokyo, Japan Person Roles submitter Protocol Name P-GSE47610-1 P-GSE47610-3 P-GSE47610-4 P-GSE47610-2 P-GSE47610-5 Protocol Description TotalGeneSignal calculated with FeatureExtraction ver 9.5.3.1 followed Agilent GeneSpring GX7.3 analysis. Signal intensity less than 0.01 were set to 0.01. No global normalization was applied for 2 reasons: 1. the input amount of totalRNA was strictly controlled as 100 ng per sample, and 2. entire labeled molecules were hybridized to each array. Normal controls and cancer samples were compared using Welch's t-test (p<0.05) and differentially expressed miRNAs with at least a 2-fold change in expression were considered to be potential biomarkers. ID_REF = VALUE = Normalized signal intensity Agilent miRNA Microarray protocol version 1.5 (December 2007). One hundred ng of total RNA was used as an input into the labeling reaction. Agilent miRNA Microarray protocol version 1.5 (December 2007). We used the Human miRNA Microarray Kit V2 (Agilent Technologies, Santa Clara, CA), which contains 20-40 features targeting each of 723 human miRNAs (Agilent design ID 019118) as annotated in the Sanger miRBase, release 10.1. The entire labeling reaction was hybridized to each array for 20 hours at 55M-0C. Total RNA including low molecular weight RNA from tissue samples was isolated using the mirVanaTM miRNA Isolation Kit (Ambion) according to the manufacturer's instructions. The quality of the RNA samples was assessed using an Agilent 2100 Bioanalyzer, and only the samples meeting the criteria of 28S/18S > 1 and RNA Integrity Number (RIN) M-bM-^IM-% 7.5 were used for all analyses. Agilent miRNA Microarray protocol version 1.5 (December 2007). Protocol Type normalization data transformation protocol labelling protocol hybridization protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name ORGANISM PART Experimental Factor Type organism part Comment[SecondaryAccession] GSE47610 Comment[GEOReleaseDate] 2013-06-04 Comment[ArrayExpressSubmissionDate] 2013-06-03 Comment[GEOLastUpdateDate] 2013-06-05 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-47610.sdrf.txt