Identification of functional circRNAs regulating ovarian follicle development in goats.

ABSTRACT

BARKGROUND Circular RNAs (circRNAs) play important regulatory roles in a variety of biological processes in mammals. Multiple birth-traits in goats are affected by several factors, but the expression and function of circRNAs in follicular development of goats are not clear. In this study, we aimed to investigate the possible regulatory mechanisms of circRNA and collected five groups of large follicles (Follicle diameter > 6 mm) and small follicles (1 mm < Follicle diameter < 3 mm) from Leizhou goats in estrus for RNA sequencing. RESULTS: RNA sequencing showed that 152 circRNAs were differentially expressed in small and large follicles. Among them, 101 circRNAs were up-regulated in large follicles and 51 circRNAs were up-regulated in small follicles. GO and KEGG enrichment analyses showed that parental genes of the differential circRNAs were significantly enriched in important pathways, such as ovarian steroidogenesis, GnRH signaling pathway, animal autophagy and oxytocin signalling pathway. BioSignal analysis revealed that 152 differentially expressed circRNAs could target 91 differential miRNAs including miR-101 family (chi-miR-101-3p, chi-miR-101-5p), miR-202 family (chi-miR-202-5p, chi-miR-202-3p),60 circRNAs with translation potential. Based on the predicted sequencing results, the ceRNA networks chicirc_008762/chi-miR-338-3p/ARHGAP18 and chicirc_040444/chi-miR-338-3p/STAR were constructed in this study. Importantly, the new gene circCFAP20DC was first discovered in goats. The EDU assay and flow cytometry results indicated that circCFAP20DC enhanced the proliferation of follicular granulosa cells(GCs). Real-time quantitative PCR and western blotting assays showed that circCFAP20DC activated the Retinoblastoma(RB) pathway and promoted the progression of granulosa cells from G1 to S phase. CONCLUSION: Differential circRNAs in goat size follicles may have important biological functions for follicular development. The novel gene circCFAP20DC activates the RB pathway, promoting the progression of GCs from G1 to S phase. This, in turn, enhances the proliferation of follicular GCs in goats.