Abstract Body: Introduction: Neonatal pig hearts exhibit remarkable regenerative capacity within two days post-birth. In this study, we propose that a comprehensive multiomics analysis encompassing circRNA, miRNA, and mRNA data from neonatal mouse hearts can unveil intricate circRNA-miRNA-mRNA relationships associated with cardiac regeneration. Our aim is to identify new novel circRNAs and miRNAs implicated in cardiomyocyte proliferation through leveraging multiomics analysis.

Methods: Utilizing neonatal mouse hearts, we conducted circRNA, miRNA, and mRNA sequencing across four different postnatal days (P1, P3, P7, and P28; n=6, encompassing both males and females). We employed integrative bioinformatics analyses across distinct experimental designs (P1 vs P3, P1 vs P7, and P1 vs P28). Back splice junctions generated by CIRCexplorer2’s parse command was utilized to annotate sequenced circRNAs as known or novel. Validation of circRNA, miRNA, and mRNA expression levels was achieved through qRT-PCR, while protein expression levels were assessed via western blotting. Fluorescence in situ hybridization (FISH) technology was employed to determine circRNA localization. The functional significance of key genes involved in cardiomyocyte proliferation was assessed via siRNA-mediated knockdown of circRNAs and miRNAs, accompanied by cell cycle assessment using bioluminescence assay and immunostaining.

Results: Intersection of differentially expressed target genes from circRNA, miRNA, and mRNA analyses at each experimental design facilitated the construction of circRNA-miRNA-mRNA regulatory networks, aiding in the development of cardiac regeneration-related regulatory networks and inference of novel relationships between circRNA, miRNA, and mRNA. Integrative bioinformatics analysis revealed significant alterations in circRNA-miRNA-mRNA expression profiles during postnatal cardiomyocyte maturation in mice. Pathway enrichment analysis identified several differentially expressed circRNAs enriched in cell division and proliferation-related pathways. qPCR and cell cycle studies identified 17 potential circRNAs associated with cardiomyocyte proliferation in mice, with knockdown of these circRNAs significantly impacting cardiomyocyte cell cycle.

Conclusion: Our findings suggest a crucial role for circRNAs in the regulation of neonatal myocardial regeneration.