Blood-based microRNA (miRNA) signatures as biomarkers have been reported for various pathologies, including cancer, neurological disorders, cardiovascular diseases, and also infections. The regulatory mechanism behind respective miRNA patterns is only partially understood. Moreover,‘‘preserved’’ miRNAs, i.e., miRNAs that are not dysregulated in any disease, and their biological impact have been explored to a very limited extent. We set out to systematically determine their role in regulatory networks by defining groups of highly-dysregulated miRNAs that contribute to a disease signature as opposed to preserved housekeeping miRNAs. We further determined preferential targets and pathways of both dysregulated and preserved miRNAs by computing multi-layer networks, which were compared between housekeeping and dysregulated miRNAs. Of 848 miRNAs examined across 1049 blood samples, 8 potential housekeepers showed very limited expression variations, while 20 miRNAs showed highly-dysregulated expression throughout the investigated blood samples. Our approach provides important insights into miRNAs and their role in regulatory networks. The methodology can be applied to systematically investigate the differences in target genes and pathways of arbitrary miRNA sets.