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. 2017 Nov 23;7(1):16143.
doi: 10.1038/s41598-017-16344-y.

Genome-wide Analysis of Circular RNAs in Prenatal and Postnatal Pituitary Glands of Sheep

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Free PMC article

Genome-wide Analysis of Circular RNAs in Prenatal and Postnatal Pituitary Glands of Sheep

Cunyuan Li et al. Sci Rep. .
Free PMC article

Abstract

Circular RNAs (circRNAs) are a class of animal non-coding RNAs and play an impor-tant role in animal growth and development. However, the expression and function of circRNAs in the pituitary gland of sheep are unclear. Transcriptome profiling of circRNAs in the pituitary gland of sheep may enable us to understand their biological functions. In the present study, we identified 10,226 circRNAs from RNA-seq data in the pituitary gland of prenatal and postnatal sheep. Reverse transcription PCR and DNA sequencing analysis confirmed the presence of several circRNAs. Real-time RT-PCR analysis showed that sheep circRNAs are resistant to RNase R digestion and are expressed in prenatal and postnatal pituitary glands. GO and KEGG enrichment analysis showed that host genes of differentially expressed circRNAs are involved in the regulation of hormone secretion as well as in several pathways related to these processes. We determined that numerous circRNAs interact with pituitary-specific miRNAs that are involved in the biologic functions of the pituitary gland. Moreover, several circRNAs contain at least one IRES element and open reading frame, indicating their potential to encode proteins. Our study provides comprehensive expression profiles of circRNAs in the pituitary gland, thereby offering a valuable resource for circRNA biology in sheep.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Identification pipeline for circRNAs.
Figure 2
Figure 2
The information on circRNAs in the sheep pituitary gland generated by deep sequencing. The structure of circRNAs in the sheep pituitary gland. Green represents intergenic regions, light red indicates exons, and light blue shows introns.
Figure 3
Figure 3
Verification of circRNAs data from RNA-seq. (a) Divergent primers used in the amplification of circular junctions. Red arrows represent divergent primers. (b) RT-PCR amplification of circRNAs with divergent primers. M is the marker (Takara DL500: 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp, and 50 bp), and N is the negative control. (c) Head-to-tail junctions were confirmed by DNA sequencing. (d) Resistance testing of circRNAs to RNase R digestion by real-time RT-PCR. β-Actin was used as a linear control. Error bars indicate ± SD.
Figure 4
Figure 4
Analysis and validation of differentially expressed circRNAs in the PG_E and PG_A groups. (a) Volcano plot analysis of all circRNAs between PG_E and PG_A group. The logarithm of the significant difference between the two samples was analyzed by log2 (fold change) as the abscissa, and the negative logarithm-log 10 (P-value) of the P value was calculated as the ordinate (P < 0.05). Red dots indicate upregulated genes; green dots represent downregulated genes. (b,c) Change in circRNA levels between the PG_E and PG_A groups. PG_E/PG_A ratios for 10 different circRNAs based on the Illumina next-generation sequencing (RNA-seq) data (one sample) (b). Expression of differentially expressed genes as determined by real-time RT-PCR (three biological replicates, each done in triplicate) (c). Error bars indicate ± SD.
Figure 5
Figure 5
Annotations and enrichment of differentially expressed circRNAs. (a) GO analysis show 792 significantly (PG_A vs. PG_E) enriched terms (P < 0.05) in the categories of biological process, molecular function, and cellular components. (b) KEGG pathway analysis identified total of 270 terms that were enriched with differentially expressed circRNAs in the PG_E and PG_A groups. The thyroid hormone signaling pathway, GnRH signaling pathway, and phosphatidylinositol signaling pathway are involved in hormone secretion regulation in the adult pituitary and indicated by blue lines.

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