Identification of Circular RNAs Regulating Cardiomyocyte Proliferation in Neonatal Pig Hearts

Little is known about the expression patterns and functions of circular RNAs (circRNAs) in the heart of large mammals. In this study, we examined the expression profiles of circRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in neonatal pig hearts. Pig heart samples collected on postnatal days 1 (P1), 3 (P3), 7 (P7) and 28 (P28) were sent for total RNA sequencing. Our data revealed a total of 7000 circRNAs in the 24 pig hearts. Pathway enrichment analysis of hallmark gene sets demonstrated that differentially expressed circRNAs are engaged in different pathways. The most significant difference was observed between P1 and the other three groups (P3, P7 and P28) in pathways related to cell cycle and muscle development. Out of the ten circRNAs that were validated through real-time quantitative polymerase chain reaction (qRT-PCR) to confirm their expression, six exhibited significant effects on cell cycle activity in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) following small interfering RNA-mediated knockdown. The circRNA-miRNA-mRNA networks were constructed to understand the potential mechanisms of circRNAs in the heart. In conclusion, our study provided a dataset for exploring the roles of circRNAs in pig hearts. In addition, we identified several circRNAs that regulate cardiomyocyte cell cycle. Overall design: To provided insights into the complex regulatory processes involving circRNAs, miRNAs, and mRNAs during postnatal heart development in newborn pigs, we performed RNA sequencing of mRNAs and noncoding RNAs including circRNAs and miRNAs using samples from neonatal pig hearts at different developmental stages (P1, P3, P7, and P28). The heart samples were sent to two different companies for sequencing. In the first batch, 12 samples from animals of different ages (3 animals at each age) were sent to Novogene Inc. for circRNA sequencing (annotated as “nv1”). A second batch of 12 more samples was sent to CD Genomocs Inc. (annotated as “cd”) for total RNA sequencing. In the third batch, new samples from the same 12 animals in the first batch were sent to Novogene Inc. (annotated as “nv2”) for total RNA sequencing. We validated the sequencing data using real-time quantitative polymerase chain reaction (qRT-PCR). Differential expression analysis and pathway enrichment analysis were done using different bioinformatics tools. Functional experiments in the cultured cardiomyocytes via small interfering RNA (siRNA) mediated knockdown of selected circRNA candidates.