Transcriptome Analysis Reveals Differentially Expressed circRNAs Associated with Fecundity in Small-Tail Han Sheep Thyroid with Different FecB Genotypes

Abstract

Litter size is an economically important trait in sheep, and it is a complex trait controlled by multiple genes in multiple organs. Among them, the regulation of lamb number trait by the thyroid gland is a very important part. However, the molecular mechanisms of the thyroid gland in sheep reproduction remain unclear. Here, RNA-seq was used to detect transcriptome expression patterns in the thyroid gland between follicular phase (FP) and luteal phase (LP) in FecB BB (MM) and FecB ++ (ww) STH sheep, respectively, and to identify differentially expressed circRNAs (DECs) associated with reproduction. Bioinformatic analysis of the source genes of these DECs revealed that they can be enriched in multiple signaling pathways involved in the reproductive process of animals. We found that the source genes of these DECs, such as GNAQ, VEGFC, MAPK1, STAT1, and HSD17B7, may play important roles in the reproductive process of animals. To better understand the function of these DECs, we constructed circRNA-miRNA co-expression networks. Dual luciferase reporter assays suggested that a ceRNA regulatory mechanism between circ_0003259-oar-miR-133-TXLNA and circ_0012128-oar-miR-370-3p-FGFR1 may hold. All of these DEC expression profiles in the thyroid gland provide a novel resource for elucidating the regulatory mechanisms underlying STH sheep prolificacy.

Keywords: CircRNA; STH sheep; follicular phase; litter size; luteal phase; thyroid gland.

Figure 1

Characterization of thyroid circRNA in sheep. Characterization of thyroid circRNA in sheep. (A) circRNA length distribution map, (B) circRNA expression box diagram.

Figure 2

DECs analysis among 4 groups. Volcano plots show the up- and down-regulation distribution of DELs in MM-FT vs. MM-LT (A), MM-FT vs. ww-FT (B), MM-LT vs. ww-LT (C), and ww-FT vs. ww-LT (D), where red and green represent up- or down-regulation, respectively. Heatmap (E) shows the expression patterns of the 4 groups of DECs.

Figure 3

Top 10 enriched GO terms of host genes of DECs in 4 groups. The top 10 enriched GO terms of host genes of DECs in (A) MM-FT vs. MM-LT (B) MM-FT vs. ww-FT, (C) MM-LT vs. ww-LT, (D) ww-FT vs. ww-LT. The horizontal and vertical coordinates represent the GO terms and -lg(p-value) of the enriched genes, respectively.

Figure 4

Twenty enriched KEGGs of host genes of DECs target genes in 4 groups. Twenty enriched KEGG of host genes of DECs in (A) MM-FT vs. MM-LT, (B) MM-FT vs. ww-FT, (C) MM-LT vs. ww-LT, (D) ww-FT vs. ww-LT. horizontal and vertical coordinates represent the -lg(p-value) of enriched genes and KEGG pathway, respectively. The rich factor is the ratio of the number of differentially expressed genes to the number of annotated genes enriched in that pathway term. The size of the circles in the graph indicates the number of differential genes enriched in the pathway.

Figure 5

circRNA–miRNA network interaction analysis. The DECs–DEMs network between (A) MM-FT vs. MM-LT, (B) MM-FT vs. ww-FT, (C) MM-LT vs. ww-LT, (D) ww-FT vs. ww-LT. Note: Nodes represent circRNA and miRNA connecting lines represent the interaction between circRNA and miRNA, red represents circRNA, and green represents miRNA. Octagon, inverted diamond, and circle represent circRNA and miRNA, respectively.

Figure 6

RT−qPCR verification of DECs. RT-qPCR verified the expression trend of DECs in (A) MM-FT vs. MM-LT, (B) MM-FT vs. ww-FT, (C) MM-LT vs. ww-LT, (D) ww-FT vs. ww-LT.

Figure 7

Plasmid construction results. (A) circ_0003259 overexpression vector. (B) circ_0012128 sequencing results. (C) TXLNA-3′UTR WT sequencing results. (D) TXLNA-3′UTR MT sequencing results. (E) FGFR1-3′UTR WT sequencing results. (F) FGFR1-3′UTR MT sequencing results.

Figure 8

Double luciferase activity detection results. (A) Relative fluorescence activity was detected after cotransfection of oar-miR-133, oar-miR-133 mimics NC, TXLNA-WT, and TXLNA-MT into 293T cells. (B) Relative fluorescence activity was detected after cotransfection of oar-miR-370-3p mimics, oar-miR-370-3p mimics NC, FGFR1-WT, and FGFR1-MT into 293T cells. (C) Relative fluorescence activity was detected after cotransfection of circ_0003259, oar-miR-133 mimics, and TXLNA into 293T cells. (D) Relative fluorescence activity was detected after cotransfection of circ_0012128, oar-miR-370-3p mimics, and FGFR1 into 293T cells. Note: “*” indicates significant differences (p > 0.05).