Dysregulated circRNA expression profile and associated miRNA sponging in abnormal lung development in congenital diaphragmatic hernia

RationaleCircular RNAs (circRNAs) can function as disease biomarkers. Their profile in abnormal lung development in congenital diaphragmatic hernia (CDH) is unknown. ObjectiveTo evaluate circRNA expression profile in CDH-associated abnormal lung development. MethodsWe profiled circRNAs in rat CDH and control lungs at embryonic day (E)15 and E21 by microarray. We validated identified circRNAs using back-splice junction amplicon sequencing, RT-qPCR, and in situ hybridization. We modified a CircRNA Function prediction Tool to predict CircRNA::micro(mi)RNA::messenger(m)RNA interactions and compared these with Oxford Nanopore RNA sequencing and existing human CDH datasets. Measurements and Main ResultsMicroarrays revealed a unique circRNA biosignature during CDH lung development. CircAnp32e was expressed in a sex-specific and spatiotemporal expression pattern in the epithelium at E15. The predicted mature sequence of circAnp32e overlapped >90% with its human orthologue. CircRNA::miRNA::mRNA interaction networks in E15 and E21 revealed enrichment in inflammation/infection, smooth muscle cell function, cell proliferation/cell cycle regulation, and response to hypoxia pathways. Parental genes of differential expressed circRNAs at E15 enriched pathways linked to cell proliferation/cell cycle/cancer, while at end-gestation, inflammation and cardiovascular processes were also overrepresented. Rat and human CDH lungs showed overlapping pathways with additional enrichment for RNA processing and protein binding/modification in humans. ConclusionA unique circRNA signature during abnormal lung development in CDH may mediate inflammatory responses, smooth-muscle-cell function, and cell proliferation regulation via miRNA sponging. Overlap of downstream pathways in rat and human CDH suggest conserved functions across species. CircRNAs may serve as biomarkers to guide prenatal management and mitigate aberrant lung development in CDH.