Identification of competing endogenous RNA networks involved in phrenic nerve stimulation preventing mechanical ventilation induced diaphragm dysfunction.

Ventilator-induced diaphragmatic dysfunction (VIDD) is characterized by diaphragmatic atrophy and contractile failure, leading to prolonged intensive care unit (ICU) stays and increases mortality. While phrenic nerve stimulation (PNS) has demonstrated efficacy in mitigating VIDD by preserving diaphragmatic activity, its underlying molecular mechanisms remain unclear. This study aimed to elucidate the role of competing endogenous RNA (ceRNA) networks in PNS-mediated protection against VIDD through integrated miRNA-Seq and RNA-Seq analyses in a rabbit model. Eleven adult male New Zealand white rabbits were divided into control (n = 4), MV (n = 3) and PNS groups (n = 4). MV and PNS groups underwent 24 h of MV, with intermittent bilateral transvenous PNS applied only to the PNS group. Differentially expressed (DE) analysis of mRNAs, miRNAs and circRNAs across pairwise group comparisons was performed via RNA-seq and miRNA-seq. Functional enrichment analyses (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) identified key pathways. Potential miRNA targets and interacting circRNAs were computationally predicted. An integrated ceRNA network was constructed using major DE RNAs to identify PNS-associated core regulators. CeRNA network was validated by quantitative real-time polymerase chain reaction (RT-qPCR) and dual-luciferase assays. High-throughput sequencing revealed significant dysregulation of miRNAs, circRNAs and mRNAs in the diaphragm following MV which was partially reversed by PNS. Bioinformatic screening identified a ceRNA network, wherein two key miRNAs emerged: miR-500-3p (targeting RAB37, an autophagy-related gene) and miR-133b-3p (targeting L-selectin, a cell adhesion molecule regulating immune responses and fibrosis). Both miRNAs were down-regulated after MV and restored by PNS. Computational prediction also identified five circRNAs (circRNA_12437, 24673, 14127, 14942, 12463) as putative sponges for these miRNAs, although this interaction lacks experimental confirmation. Dual-luciferase assays confirmed direct binding of miR-500-3p to RAB37 and miR-133b-3p to L-selectin, functionally linking them to PNS-mediated VIDD protection. Enrichment analyses indicated that DE genes were predominantly enriched in phagosome activity and cell adhesion molecule pathways. Collectively, these findings suggest that PNS preserves diaphragmatic function by modulating ceRNA networks to suppress excessive autophagy and immune cell infiltration. This study identifies the first PNS-responsive ceRNA network in VIDD pathogenesis. Our data highlight the potential critical roles of miR-500-3p-RAB37 and miR-133b-3p-L-selectin axes in regulating autophagy and immune responses. These results provide mechanistic insights and suggest potential therapeutic targets for diaphragm dysfunction.