Dynamic expression and functional analysis of circRNA in granulosa cells during follicular development in chicken

Abstract

Background: Circular RNA (circRNA) is a type of noncoding RNA involved in a variety of biological processes, especially in post-transcriptional regulation. The granulosa cells of follicles play a determining role in ovarian development. However, the function of circRNA in chicken follicles is unclear. To better understand the molecular mechanism underlying follicular development and granulosa cell function, we performed a strategy of second-generation sequencing and linear RNA depletion for granulosa cells from small yellow follicles (SYF, 5-8 mm), the smallest hierarchal follicles (F6, 9-12 mm), and the largest hierarchal follicles (F1, ~ 40 mm).

Results: We predicted a total of 11,642 circRNAs that distributed on almost all chromosomes. The majority of the splice lengths of circRNAs were 200-500 nt and mainly produced from intron and CDS regions. During follicle growth, differentially expressed (DE) circRNAs showed dynamic changes which were tissue- and stage-specific. The host genes of DE circRNAs were functionally enriched in GTPase activity and several pathways involved in reproduction. Moreover, bioinformatic prediction analysis for circRalGPS2 demonstrated that circRNAs from the same genes may share common miRNA to act as a sponge. The predicted target genes were enriched in various biological processes including cognition, cell communication, and regulation of signaling, and several pathways related to reproduction such as tight junction, oocyte meiosis, progesterone-mediated oocyte maturation, and GnRH signaling.

Conclusions: This study provides a starting point for further experimental investigations into chicken circRNAs and casts a light on the understanding of follicle development.

Keywords: Chicken; Circular RNA; Follicles; Granulosa cells; Pathways.

Fig. 1

Overview of sequencing data of circRNA by Circos plots. Note: Plot inside are chromosomes from 1 to 28 and sex chromosome converged across granulosa cells (GCs) from SYF (G1, G2, G3), F6 (G1, G2, G3) to F1 (G1, G2, G3), SYF: small yellow follicle; F6: the smallest hierarchal follicles; F1: the largest hierarchal follicle; G: granulosa cell

Fig. 2

Profiling of circular RNAs in chicken follicle granulosa cells. Note: (a), splice length of circRNAs; (b), genomic distance between circRNAs; (c), source of circRNAs; (d), exon numbers; (e), length of host genes; (f), flank intron length distributed in granulosa cells

Fig. 3

Features of differential expression circRNAs. Note: (a), Venn analysis of differentially expressed circRNAs in three stages. b, volcano map of differentially expressed circRNAs (DEcircs) between SYF and F6 granulosa cells. c, volcano map of DEcircs between SYF and F1 granulosa cells. d, volcano map of DEcircs between F6 and F1 granulosa cells. SYF: small yellow follicle; F6: the smallest hierarchal follicles; F1: the largest hierarchal follicle; G: granulosa cell

Fig. 4

Heat map representing the relative expression levels of all differentially expressed circRNAs in the three stages (Fold change > 2, P < 0.05). Note: SYF: small yellow follicle; F6: the smallest hierarchal follicles; F1: the largest hierarchal follicle; G: granulosa cell.

Fig. 5

GO and KEGG analysis for the host genes of all differentially expressed circRNAs. Note: (a), The top 10 GO enrichment term in BP, CC, and MF for the host genes of all differentially expressed circRNAs; (b), the top 20 KEGG enrichment term for the host genes of all differentially expressed circRNAs

Fig. 6

Differentially expressed circRNAs in three different stages of granulosa cells. Note: a, Venn diagram of differentially expressed circRNAs (n = 891, fold change > 2, q-value < 0.05); b, Heat map of pervasive circRNAs shows expression value at three stages. SYF: small yellow follicle; F6: the smallest hierarchal follicles; F1: the largest hierarchal follicle; G: granulosa cells.

Fig. 7

Validation of circRNAs by experimental and sequencing. Note: (a), agarose gel electrophoresis test for divergent primers and convergent primers amplify circRNAs; The brightest DNA marker is 200 bp. cc: convergent primers for cDNA; cg: convergent primers for genomic DNA; dc: divergent primers for cDNA; dg: divergent primers for genomic DNA. b, Sanger sequencing confirmed the back-splicing junction of circRNAs; (c), RT-qPCR validation of nine differentially expressed circRNAs in three types of granulosa cell. TPM: Transcripts Per Million; RQ: relative expression; Graph number of a, b, c, d, e, f, g, h, i represent circRNAs: circRNA_8: 6369673|6,402,248, circRNA_8: 6369673|6,422,097, circRNA_8: 6384248|6,402,248, circRNA_26: 4587446|4,664,529, circRNA_3: 66399375|66,402,723, circRNA_20: 2426006|2,445,902, circRNA_14: 84666291|8,458,456, circRNA_12: 9000575|9,001,598, circRNA_23: 5084518|5,086,817, respectively. SYF: small yellow follicle; F6: the smallest hierarchal follicles; F1: the largest hierarchal follicle; G: granulosa cell.

Fig. 8

Expression pattern of circRLAGPS2 during ovarian development Note: F1, F2, F3, F4, F5, F6 represent hierarchal follicles from larger to small; SYF, small yellow follicle; O, ovary; LWF, large white follicle, POF, post-ovulation follicle; Ut, uterus; Ov, oviduct.

Fig. 9

Predicted biomathematical circRNA–miRNA network for the same soured circRNAs. Note: Pink square represents circRNAs, blue circle represents miRNAs.

Fig. 10

GO and KEGG analysis of circRalGPS2-miRNA target genes. Note: (a), Gene ontology of target genes. b, KEGG pathways of target genes.