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.

Keywords: Follicular development; Leizhou goat; RNA sequencing; Reproduction; circCFAP20DC.

Fig. 1

Identification of follicular circRNAs in goats. A, Length distribution of circRNAs. B, Chromosomal distribution of circRNAs. C, Types of reverse splicing of circRNAs. D, Distribution of circRNAs expression

Fig. 2

Differential expression of circRNA in small and large follicles. A, Volcano map of differentially expressed circRNAs. B, Clustering map of differentially expressed circRNAs

Fig. 3

Functional enrichment analysis of parental genes for circRNAs. A, GO enrichment analysis of the first 20 pathways. B, The first ten pathways of biological processes, cellular components and molecular functions in GO enrichment analysis. C. Top 20 terms for KEGG enrichment analysis. D. Bar graph of significantly enriched pathways in KEGG analysis

Fig. 4

Prediction and quantitative validation of circRNAs function. A, CeRNA network construction. Orange is circRNA, cyan is miRNA, blue is mRNA; arrows facing down are downregulated in small follicles, arrows facing up are upregulated in small follicles. B, Prediction of circRNA translation potential. IRES is dependent on the presence of an internal ribosome entry site to initiate translation, and m6A is dependent on the presence of N6-methyladenosine residues to initiate translation. C, QPCR validation of differential expression of circRNAs in sequencing data

Fig. 5

Identification of circCFAP20DC. A, Convergent primer, divergent primer amplification. B, CircCFAP20DC full-length amplification. C, Nucleoplasmic separation to verify the subcellular localisation of circCFAP20DC and GAPDH as a localised cytoplasmic control. D, Ring-forming mechanism of circCFAP20DC. E, RNase R digestion assay to validate the stability of circCFAP20DC. F, Comparison of circCFAP20DC full-length sequences, Query is sequenced sequence, Sbjct is amplified sequence

Fig. 6

Phenotypic effects of circCFAP20DC overexpression on goat granulosa cells. A, EDU detects the proliferative effect of circCFAP20DC overexpression on goat granulosa cells. B, Effect of circCFAP20DC overexpression on the cell cycle of goat granulosa cells detected by flow cytometry

Fig. 7

QPCR detection of PCNA(A), MCM4(B), MCM6(C), MCM7(D), CCNE1(E), CCNE2(F), CDK2(G), E2F1(H), E2F2(I) expression levels after circCFAP20DC overexpression

Fig. 8

Changes in protein levels after overexpression of circCFAP20DC. A, Changes in PCNA and MCM6 at the protein level. B, Changes in cell cycle-related genes at the protein level