A pilot study of peripheral blood tsRNA expression profiling in children with influenza-associated acute necrotizing encephalopathy

Influenza-associated acute necrotizing encephalopathy (ANE) is a fulminant pediatric neurological complication primarily driven by hypercytokinemia. Given the lack of specific biomarkers, early diagnosis remains clinically challenging. This study aimed to characterize the peripheral blood expression profiles of tRNA-derived small RNAs (tsRNAs) in pediatric ANE to identify novel non-invasive diagnostic markers and therapeutic targets. Nine pediatric subjects (n = 3 per group) were enrolled and stratified into three cohorts: ANE (severe), uncomplicated influenza (mild), and healthy controls (normal). Peripheral blood tsRNA expression was profiled utilizing high-throughput microarrays. Comprehensive bioinformatic analyses—including principal component analysis (PCA), Venn intersection analysis, GO, and KEGG—were conducted to evaluate transcriptomic patterns and biological functions of differentially expressed tsRNAs (DE-tsRNAs). Microarray results were technically validated by qRT-PCR on the same sample cohort. Unsupervised PCA successfully delineated distinct transcriptomic clusters correlating with disease severity, identifying > 200 ANE-specific DE-tsRNAs. qRT-PCR validation confirmed the significant upregulation of tRF5-24-LysTTT-3 and tRF5-25-ProTGG-2, and the downregulation of 5’tiRNA-35-GluTTC-9 and 5’tiRNA-36-GluTTC-7. Bioinformatic analysis predicted that the target genes of these dysregulated tsRNAs are predominantly implicated in neurogenesis, the RAS signaling pathway, and autophagy. This pilot study provides the first evidence of systemic tsRNA dysregulation in pediatric influenza-associated ANE. Importantly, comparative analysis suggests this distinct transcriptomic signature may represent a highly specific molecular surrogate of ANE neuropathogenesis, rather than a generalized systemic inflammatory response. These findings offer novel insights into specific neuroinflammatory cascades and highlight tsRNAs as promising disease-specific biomarkers, warranting further validation in larger, independent cohorts.