Circulating microRNAs in preterm white matter injury: a systems biology/qPCR-based pilot study

BACKGROUND: White matter injury (WMI) is a major cause of neurodevelopmental impairment in preterm infants (PTIs), yet early molecular biomarkers remain elusive. Circulating microRNAs (miRNAs) hold promise, but neonatal biosample limitations challenge the use of high-throughput methods like next-generation sequencing (NGS). We evaluated the feasibility of quantitative PCR (qPCR) as a primary discovery tool for circulating miRNA biomarkers of WMI, supported by systems biology modeling and selective NGS validation. METHODS: Plasma-derived miRNAs from PTIs with and without WMI were profiled using qPCR after stringent hemolysis screening. Candidate miRNAs were curated from brain-specific regulatory networks. ROC bootstrapping and gene ontology/pathway enrichment were used to assess diagnostic and functional relevance. Boolean logic modeling simulated miRNA-mediated regulation of oligodendrocyte maturation. RESULTS: miR-23a-3p and miR-17-5p were differentially expressed across WMI states and showed moderate discriminative power (AUCs: 0.71 and 0.68), while oligodendrocyte miRNAs (miR-219a-2-3p, miR-338-5p) were consistently low. Boolean simulations confirmed miR-23a and miR-17 modulate myelination via PTEN repression and PI3K/Akt pathway activation. qPCR and model predictions aligned strongly; NGS showed discordant trends likely due to detection biases. CONCLUSION: This pilot study demonstrates that qPCR, when combined with systems modeling, proposes a viable and sensitive discovery tool for miRNA biomarkers in clinically constrained populations. IMPACT: This study demonstrates that qPCR combined with systems biology modeling, is a viable and biologically coherent approach for identifying circulating miRNA biomarkers of white matter injury in preterm infants. It challenges the conventional reliance on next-generation sequencing as the default discovery platform, and repositions qPCR as a powerful primary discovery method for diagnostic miRNA biomarkers when coupled with systems biology modeling, showing that qPCR can yield translational insights in ethically and clinically constrained neonatal populations. By integrating Boolean logic simulations, the study links key miRNAs (miR-23a-3p and miR-17-5p) to mechanistic pathways of oligodendrocyte maturation, offering a functional layer to biomarker discovery.