Identification and Profiling of Pituitary microRNAs of Sheep during Anestrus and Estrus Stages.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, molecules of 21 to 25 nucleotides in length, that regulate gene expression by binding to their target mRNA and play a significant role in animal development. The expression and role of miRNAs in regulating sheep estrus, however, remain elusive. Transcriptome analysis is helpful to understand the biological roles of miRNAs in the pituitary gland of sheep. A sheep's pituitary gland has a significant difference between estrus and anestrus states. Here, we investigate the expression profiles of sheep anterior pituitary microRNAs (miRNAs) in two states, estrus and anestrus, using Illumina HiSeq-technology. This study identified a total of 199 miRNAs and 25 differentially expressed miRNAs in the estrus and anestrus pituitary gland in sheep. Reverse transcription quantitative-PCR (RT-qPCR) analysis shows six differentially (p < 0.05) expressed miRNAs, that are miR-143, miR-199a, miR-181a, miR-200a, miR-218, and miR-221 in both estrus and anestrus states. miRNAs containing estrus-related terms and pathways regulation are enriched using enrichment analysis from gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Moreover, we also envisioned a miRNA-mRNA interaction network to understand the function of miRNAs involved in the pituitary gland regulatory network. In conclusion, miRNA expression profiles in sheep pituitary gland in the anestrus and estrus deliver a theoretical basis for the study of pituitary gland biology in sheep.

Analysis of pituitary transcriptomics indicates that lncRNAs are involved in the regulation of sheep estrus.

Seasonal estrus is a key factor limiting animal fertility, and understanding the molecular mechanisms that regulate animal estrus is important for improving animal fertility. The pituitary gland, which is the most important endocrine gland in mammals, plays an important role in regulating the physiological processes such as growth, development, and reproduction of animals. Here, we used RNA-seq technology to study the expression profile of lncRNAs in the anterior pituitary of sheep during estrus and anestrus. In this study, we identified a total of 995 lncRNAs, of which 335 lncRNAs were differentially expressed in two states (including 38 up-regulated and 297 down-regulated lncRNAs). RT-qPCR verified the expression levels of several lncRNAs. Target predictive analysis revealed that these lncRNAs can act in cis or trans and regulate the expression of genes involved in the regulation of sheep estrus. Target gene enrichment analysis of differentially expressed lncRNAs indicates that these lncRNAs can regulate sheep estrus by regulating hormone metabolism and energy metabolism. Through our research, we provide the expression profile of lncRNAs in the pituitary of sheep, which provides a valuable resource for further understanding of the genetic regulation of seasonal estrus in sheep from the perspective of lncRNAs.

Circ-HIPK3 plays an active role in regulating myoblast differentiation.

The development of skeletal muscle is a complex biological process involving a variety of regulatory pathways. A deeper understanding of the developmental mechanisms of skeletal muscle is of great significance for the treatment of muscular lesions. In recent years, studies have shown that circRNAs, as a new class of post-transcriptional regulators, play an important regulatory role in a variety of biological processes, but their function in skeletal muscle development remains unclear. Here, we used C2C12 myoblasts to study circ-HIPK3, which has significant differences in the developmental stages of skeletal muscle and is highly expressed. We found that the expression level of circ-HIPK3 was significantly up-regulated (p < .05) with the persistence of C2C12 cell differentiation. Combining the results of bioinformatics analysis and dual luciferase reporter experiments, we found that circ-HIPK3 is a sponge of miR-124 and miR-379 that regulate muscle differentiation. Our study shows that circ-HIPK3 can promote the differentiation of C2C12 myoblasts, providing a scientific basis for further research on the development of skeletal muscle at the level of circRNAs.

Circular RNA circ-FoxO3 Inhibits Myoblast Cells Differentiation.

CircRNA is a type of closed circular non-coding RNA formed by reverse splicing and plays an important role in regulating the growth and development of plants and animals. To investigate the function of circ-FoxO3 in mouse myoblast cells' (C2C12) differentiation and proliferation, we used RT-qPCR to detect the expression level of circ-FoxO3 in mouse myoblast cells at different densities and different differentiation stages, and the specific interference fragment was used to inhibit the expression level of circ-FoxO3 in myoblast cells to observe its effect on myoblast cells proliferation and differentiation. We found that the expression level of circ-FoxO3 in myoblast cells increased with the prolongation of myoblast cells differentiation time, and its expression level decreased with the proliferation of myoblast cells. At the same time, we found that the differentiation ability of the cells was significantly increased (p < 0.05), but the cell proliferation was unchanged (p > 0.05) after inhibiting the expression of circ-FoxO3 in myoblast cells. Combining the results of bioinformatics analysis and the dual luciferase reporter experiment, we found that circ-FoxO3 is a sponge of miR-138-5p, which regulates muscle differentiation. Our study shows that circ-FoxO3 can inhibit the differentiation of C2C12 myoblast cells and lay a scientific foundation for further study of skeletal muscle development at circRNA levels.

Expression profiles of microRNAs in skeletal muscle of sheep by deep sequencing.

OBJECTIVE: MicroRNAs are a class of endogenous small regulatory RNAs that regulate cell proliferation, differentiation and apoptosis. Recent studies on miRNAs are mainly focused on mice, human and pig. However, the studies on miRNAs in skeletal muscle of sheep are not comprehensive. METHODS: RNA-seq technology was used to perform genomic analysis of miRNAs in prenatal and postnatal skeletal muscle of sheep. Targeted genes were predicted using miRanda software and miRNA-mRNA interactions were verified by quantitative real-time polymerase chain reaction. To further investigate the function of miRNAs, candidate targeted genes were enriched for analysis using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment. RESULTS: The results showed total of 1,086 known miRNAs and 40 new candidate miRNAs were detected in prenatal and postnatal skeletal muscle of sheep. In addition, 345 miRNAs (151 up-regulated, 94 down-regulated) were differentially expressed. Moreover, miRanda software was performed to predict targeted genes of miRNAs, resulting in a total of 2,833 predicted targets, especially miR-381 which targeted multiple muscle-related mRNAs. Furthermore, GO and KEGG pathway analysis confirmed that targeted genes of miRNAs were involved in development of skeletal muscles. CONCLUSION: This study supplements the miRNA database of sheep, which provides valuable information for further study of the biological function of miRNAs in sheep skeletal muscle.

Genome-wide analysis of circular RNAs in prenatal and postnatal muscle of sheep.

Circular RNAs (circRNAs), a type of non-coding RNA with circular structure, were generated by back splicing and widely expressed in animals and plants. Recent studies have shown that circRNAs extensively participate in cell proliferation, cell differentiation, cell autophagy and other biological processes. However, the role and expression of circRNAs in the development and growth of muscle have not been studied in sheep. In our study, we first used RNA-seq to study the circRNAs in prenatal and postnatal longissimus dorsi muscle of sheep. A total of 6113 circRNAs were detected from the RNA-seq data. Several circRNAs were identified using reverse transcription PCR, DNA sequencing and RNase R digestion experiments. The expression levels of several circRNAs in prenatal and postnatal muscle were confirmed by Real-Time RT-PCR. The gene ontology (GO) and KEGG enrichment analysis of the host gene of the circRNAs showed that these circRNAs were mainly involved in the growth and development of muscle related signaling pathways. These circRNAs might sponge microRNAs (miRNAs) in predicted circRNA-miRNA-mRNA networks. The circRNAs expression profiles in muscle provided an important reference for the study of circRNAs in sheep.

Genome-wide analysis of circular RNAs in prenatal and postnatal pituitary glands of sheep.

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.

Aberrant expression of miR-127, miR-21 and miR-16 in placentas of deceased cloned sheep.

Placental deficiencies are associated with developmental abnormalities of animal produced by somatic cell nuclear transfer (SCNT). It is reported that aberrant expression of microRNAs (miRNAs) in the common placenta is associated with fetal growth restriction and placental deficiencies. However, an understanding of the expression and function of miRNAs in the placentas of cloned animal is lacking. In this study, we characterized the expression of five growth-associated miRNAs (miR-127, miR-16, miR-21, miR-93 and miR-182) in placentas of deceased transgenic cloned sheep (deceased group, n=7), live transgenic cloned sheep (live group, n=5) and conventionally produced sheep (control group, n=10). Expression levels of miR-127 (P<0.01), miR-21 (P<0.01) and miR-16 (P<0.05) were significantly up-regulated in the placentas of deceased group compared to that of control group. In contrast, the expression of these miRNAs was largely normal in the placentas of live group, except for the expression of miR-21. Furthermore, we confirmed that retrotransposon-like gene (Rtl1), a key gene in placental development, was down-regulated by miR-127 as a target in placenta cells. Our results suggested that the abnormal expression of miR-127, miR-21 and miR-16 in placentas of deceased sheep, through dysregulation of target genes, may result in developmental deficiencies of transgenic cloned sheep.