Time-dependent miRNA regulation of molecular pathways in hepatocytes during prolonged static cold storage

Static cold storage (SCS) is limited by cold-induced ischemic injury, which restricts long-term liver preservation and adversely affects transplantation outcomes. To extend preservation duration, it is essential to characterize the molecular pathways and cellular networks activated during prolonged cold stress, including potential miRNA-mediated regulation, to elucidate injury mechanisms associated with extended SCS. To dive deeper into the molecular underpinnings of cold ischemia, we quantified the expression of 84 microRNAs (miRNAs) in isolated primary rat hepatocytes, stored for short (1hr, SCS1), moderate (24hrs, SCS2) and prolonged (72hrs, SCS3) durations. From these expression profiles, we assessed the effect of miRNAs-mediated regulation on functional enrichment using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), along with imaging to assess cell viability. Our results reveal a time-dependent influence of miRNAs on both KEGG pathways and GO terms, including biological processes (GOBP) and molecular functions (GOMF). During early stress (SCS1), miRNA-mediated inhibition was associated with GO terms related to membrane organization and energy conservation. Under moderate stress (SCS2), the response shifted toward modulation of translation and stress signaling, including pro- and anti-apoptotic pathways. Notably, SCS3 was the only group showing significant KEGG enrichment compared with SCS1, highlighting miRNA-mediated regulation of global metabolic networks and ribosome assembly. Ultimately, at SCS3, these changes reflect a multilevel collapse of cytoprotective and homeostatic mechanisms, coinciding with severe cellular damage (<5% viability). Collectively, these findings reveal dynamic miRNA regulation during cold storage and provide mechanistic insight into pathways that may be leveraged to extend organ preservation.