Bioinformatics analysis of Gene Expression Omnibus (GEO) dataset

A survey on the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/gds) repository was made to identify the publicly available miRNA expression datasets associated with RMS patients. The miRNA microarray-based expression data matrix from a cohort of 49 RMS Formalin-fixed paraffin-embedded tissues (primary not pre-treated tumors) collected at Fondazione IRCCS Istituto Nazionale dei Tumori (Milan, Italy) was retrieved for further analysis (ID: GSE135518). In this dataset, miRNA profile was performed using a SurePrint G3 Human miRNA r21 microarrays (Agilent) designed on miRBase 21.0 (miRNA). GSE135518 includes 27 pediatric RMS (0–14 years) and 22 AYA RMS (15- + 30 years)] as well as 13 normal tissue counterparts (CTRL) [27]. Primary data were collected using Agilent’s Feature Extraction software v10.7 (Agilent Technologies), background corrected, and quantile normalized using Bioconductor limma implementation in R. Differentially expressed miRNAs in the tissue sample between ARMS, ERMS and CTRL were identified imposing log2|FC|> 2 and adjusted p < 0.05 by using the GEO2R bioinformatics tool. Expression of miR-335-5p was retrieved from the data matrix and median expression was used to stratify patients for Kaplan–Meier analysis having overall survival as clinical endpoint. Patients’ clinical information’s are shown in Supplementary Table 2.