Whole transcriptome analysis revealed the regulatory network and related pathways of non-coding RNA regulating ovarian atrophy in broody hens

Abstract

Poultry broodiness can cause ovarian atresia, which has a detrimental impact on egg production. Non-coding RNAs (ncRNAs) have become one of the most talked-about topics in life sciences because of the increasing evidence of their novel biological roles in regulatory systems. However, the molecular mechanisms of ncRNAs functions and processes in chicken ovarian development remain largely unknown. Whole-transcriptome RNA sequencing of the ovaries of broodiness and laying chickens was thus performed to identify the ncRNA regulatory mechanisms associated with ovarian atresia in chickens. Subsequent analysis revealed that the ovaries of laying chickens and those with broodiness had 40 differentially expressed MicroRNA (miRNAs) (15 up-regulated and 25 down-regulated), 379 differentially expressed Long Noncoding RNA (lncRNAs) (213 up-regulated and 166 down-regulated), and 129 differentially expressed circular RNA (circRNAs) (63 up-regulated and 66 down-regulated). The competing endogenous RNAs (ceRNA) network analysis further revealed the involvement of ECM-receptor interaction, AGE-RAGE signaling pathway, focal adhesion, cytokine-cytokine receptor interaction, inflammatory mediator regulation of TRP channels, renin secretion, gap junction, insulin secretion, serotonergic synapse, and IL-17 signaling pathways in broodiness. Upon further analysis, it became evident that THBS1 and MYLK are significant candidate genes implicated in the regulation of broodiness. The expression of these genes is linked to miR-155-x, miR-211-z, miR-1682-z, gga-miR-155, and gga-miR-1682, as well as to the competitive binding of novel_circ_014674 and MSTRG.3306.4. The findings of this study reveal the existence of a regulatory link between non-coding RNAs and their competing mRNAs, which provide a better comprehension of the ncRNA function and processes in chicken ovarian development.

Keywords: broodiness; ceRNA network; ncRNAs; ovarian development; whole transcriptome analysis.

Figure 1

Anatomical morphological observation of ovarian tissue in poultry was conducted by examining two hematoxylin and eosin stained sections. (A) Normal ovarian tissue that continuously lays eggs. (B) Atrophy of ovaries for 30 days of broodiness. (C) Histological characteristics in egg-laying hens (staining at 200 μm). (D) Histological characteristics in egg-laying hens (staining at 50 μm). (E) Histological characteristics in broody hens (staining at 200 μm). (F) Histological characteristics in broody hens (staining at 50 μm). (G) The mitochondrial ultrastructure of the chicken egg-laying ovaries were analyzed by TEM. (H) The mitochondrial ultrastructure of the chicken broody ovaries were analyzed by TEM.

Figure 2

Overview of small RNA sequencing in the chicken ovary. (A) The length distribution of small RNA was AO1, AO2 and AO3, respectively. (B) The length distribution of small RNA was NO1, NO2 and NO3. (C) Statistical analysis of miRNA sequence abundance across various categories in each sample. (D) Statistical graph depicting the distribution of miRNA sequence species across various sample categories. exist_mirna (The miRNA of this species has been included in miRbase). known_miRNA (The identified miRNAs were compared with the known animal miRNAs in miRbase). novel_mirna (Combined with the reference sequence, the issuer structure was predicted and the miRNA was identified.) unann (The tags that cannot be annotated as any of the above molecules were recorded as unann). (E) Heatmap of differentially expressed miRNAs. (F) Compare the group NO –vs. -AO scatter plots.

Figure 3

GO and KEGG analysis of DE miRNAs. (A) The GO enrichment classification histogram of DEmiRNA is divided into three levels: BP, MF, and CC. (B) Top 20 significantly changed GOs of DEmiRNAs in biological processes. (C) The top 20 pathways significantly associated with differentially expressed miRNA transcripts.

Figure 4

Overview of lncRNA sequencing in the chicken ovary. (A) CPC2, CNCI, and Feelnc were used to evaluate the encoding capabilities of all transcripts. The intersection of transcripts without coding potential is a reliable prediction result. (B) Total lncRNA type statistics. (C) lncRNA expression abundance distribution map. (D) Violin diagram of lncRNA expression. (E) Comparison of group NO-vs. -AO volcano maps. (F) Compare group NO-vs. -AO heat maps.

Figure 5

Overview of circRNA sequencing in the chicken ovary. (A) All the circular RNA chromosome statistics. (B) All the circular RNA length distribution statistics. (C) Statistical map of circular RNA type distribution. (D) CircRNA statistical map of differences. (E) Comparison of group NO-vs. -AO volcano maps. (F) Differential circRNA clustering heat map.

Figure 6

CeRNA network constructed by DEmRNAs, DElncRNAs, DEcircRNAs, and DEmiRNAs. (A) mRNA-miRNA-circRNA network. (B) mRNA-miRNA-lncRNA network. The shades of the colors indicate the upregulated (red) and downregulated (green) level. The characters of the figures correspond to different RNA species, round -mRNA, diamond-miRNA, v-circRNA, triangle-lncRNA.

Figure 7

lncRNA/circRNA-miRNA-mRNA co-regulatory network. The characters of the figures correspond to different RNA species, round -mRNA, diamond -miRNA, to v-circRNA, triangle -lncRNA.

Figure 8

Validation of RNA-seq data using real time quantitative PCR (RT-qPCR).