# Validation of Targeted Relationships of Novel circRNA803/lncRNA MSTRG.19726–oar-let-7a–CPEB1 ceRNA Networks, Key to Follicle Development in Single-Litter and Multi-Litter Sheep Based on Whole-Transcriptome Sequencing

## TL;DR

This study identifies a key RNA regulatory network involved in sheep follicle development, which could help improve sheep reproductive efficiency.

## Contribution

The novel ceRNA network involving circRNA803, lncRNA MSTRG.19726, oar-let-7a, and CPEB1 is validated as critical for follicle development in sheep.

## Key findings

- A ceRNA network involving circRNA803, lncRNA MSTRG.19726, oar-let-7a, and CPEB1 was identified and validated.
- oar-let-7a regulates CPEB1 expression and is linked to reproductive signaling pathways like oocyte meiosis and Hippo.
- qRT-PCR confirmed RNA-seq results, supporting the reliability of the identified regulatory mechanisms.

## Abstract

The objective of this study is to investigate the molecular regulatory mechanisms of non-coding RNA (ncRNA) during the developmental process of multi-litter sheep ovaries and identify key regulatory genes that enhance the reproductive capacity of sheep. This study selected Small-Tail Han sheep (multi-litter sheep) and Ujumuqin sheep (single-litter sheep) as comparative models, constructed the expression profiles of ncRNAs and mRNAs in ovarian tissues, identified differentially expressed (DE) lncRNAs, circRNAs, miRNAs, and mRNAs, and performed target gene prediction along with functional and signaling pathway enrichment analyses. Reproduction-related pathways were further screened to construct competing endogenous RNA (ceRNA) regulatory networks (lncRNA–miRNA–mRNA and circRNA–miRNA–mRNA). Finally, the dual-luciferase reporter gene assay system was employed to perform the functional validation of the relevant targeted regulatory effects. A comprehensive screening identified 411 DE lncRNAs, 322 DE circRNAs, 26 DE miRNAs, and 29 DEGs from the ovarian tissues of Ujumqin and Small-Tail Han sheep. The results of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses demonstrated that the DE target genes were significantly enriched in pathways associated with cell dedifferentiation, the positive regulation of embryonic development, glycosaminoglycan biosynthesis, Hippo signaling, and other signaling pathways. To identify genes associated with reproductive processes, we performed differential expression screening followed by pathway enrichment analysis, which revealed significant enrichment in reproductive regulatory pathways. Based on these findings, we constructed a ceRNA regulatory network incorporating 22 DEGs, 17 DE lncRNAs, three DE circRNAs, and one DE miRNA. Our analysis revealed that oar-let-7a is involved in signaling pathways such as oocyte meiosis and Hippo, suggesting it may serve as a key miRNA regulating the trait of multiple offspring. The dual-luciferase reporter assay was employed to confirm that oar-let-7a directly targets and regulates the expression of CPEB1. Additionally, it was demonstrated that circRNA803 and lncRNA MSTRG.19726 function as molecular sponges to competitively bind and regulate oar-let-7a. These findings suggest that oar-let-7a mediates the expression of CPEB1 via circRNA803 and lncRNA MSTRG.19726 sponge adsorption, thereby regulating the process of follicular dominance in sheep. The qRT-PCR method was employed to validate the expression patterns of nine randomly selected DEGs, and the results corroborated the reliability of the RNA-seq sequencing data. This study investigated the coordinated regulatory mechanism of DE ncRNAs and their corresponding target genes, identifying a ceRNA network, circRNA803/lncRNA MSTRG.19726-oar-let-7a-CPEB1, which plays a critical role in regulating the process of follicular dominance in sheep. These findings provide fundamental data for uncovering the reproductive potential of sheep and facilitate a comprehensive understanding of their reproductive characteristics, which hold significant guiding implications for enhancing reproductive efficiency.