Functional analysis of differentially expressed circular RNAs in sheep subcutaneous fat.

BACKGROUND: Circular RNAs (circRNAs), as important non-coding RNAs (ncRNAs), are involved in many biological activities. However, the exact chemical mechanism behind fat accumulation is unknown. In this paper, we obtained the expression profiles of circRNAs using high-throughput sequencing and investigated their differential expression in subcutaneous fat tissue of Duolang and Small Tail Han sheep. RESULTS: From the transcriptomic analysis, 141 differentially expressed circRNAs were identified, comprising 61 up-regulated circRNAs and 80 down-regulated circRNAs. These host genes were primarily enriched in the MAPK and AMPK signaling pathways which is closely associated with fat deposition regulation. We identified circRNA812, circRNA91, and circRNA388 as vital genes in fat deposition by miRNA-circRNA target gene prediction. The functional annotation results of target genes of key circRNAs showed that the signaling pathways mainly included PI3K-Akt and AMPK. We constructed the competing endogenous RNA (ceRNA) regulatory network to study the role of circRNAs in sheep lipid deposition, and circRNA812, circRNA91, and circRNA388 can adsorb more miRNAs. NC_040253.1_5757, as the source of miRNA response element (MRE) among the three, may play an important role during the process of sheep fat deposition. CONCLUSIONS: Our study gives a systematic examination of the circRNA profiles expressed in sheep subcutaneous fat. These results from this study provide some new basis for understanding circRNA function and sheep fat metabolism.

Genome wide identification and characterization of fertility associated novel CircRNAs as ceRNA reveal their regulatory roles in sheep fecundity.

Reproductive traits play a vital role in determining the production efficiency of sheep. Maximizing the production is of paramount importance for breeders worldwide due to the growing population. Circular RNAs (circRNAs) act as miRNA sponges by absorbing miRNA activity through miRNA response elements (MREs) and participate in ceRNA regulatory networks (ceRNETs) to regulate mRNA expression. Despite of extensive research on role of circRNAs as miRNA sponges in various species, their specific regulatory roles and mechanism in sheep ovarian tissue are still not well understood. In this study, we performed whole genome sequencing of circRNAs, miRNA and mRNA employing bioinformatic techniques on ovine tissues of two contrasting sheep breeds "Small tail Han (X_LC) and Dolang sheep (D_LC)", which results into identification of 9,878 circRNAs with a total length of 23,522,667 nt and an average length of 2,381.32 nt. Among them, 44 differentially expressed circRNAs (DECs) were identified. Moreover, correlation between miRNA-mRNA and lncRNA-miRNA provided us with to prediction of miRNA binding sites on nine differentially expressed circRNAs and 165 differentially expressed mRNAs using miRanda. miRNA-mRNA and lncRNA-miRNA pairs with negative correlation were selected to determine the ceRNA score along with positively correlated pairs from lncRNA and mRNA network. Integration of ceRNA score and positively correlated pairs exhibit a significant ternary relationship among circRNAs-miRNA-mRNA demonestrated by ceRNA, comprising of 50 regulatory pairs sharring common nodes and predicted potential differentially expressed circRNAs-miRNAs-mRNAs regulatory axis. Based on functional enrichment analysis shortlisted key ceRNA regulatory pairs associated with reproduction including circRNA_3257-novel579_mature-EPHA3, circRNA_8396-novel130_mature-LOC101102473, circRNA_4140- novel34_mature > novel661_mature-KCNK9, and circRNA_8312-novel339_mature-LOC101110545. Furthermore, expression profiling, functional enrichments and qRT-PCR analysis of key target genes infer their implication in reproduction and metabolism. ceRNA target mRNAs evolutionary trajectories, expression profiling, functional enrichments, subcellular localizations following genomic organizations will provide new insights underlying molecular mechanisms of reproduction, and establish a solid foundation for future research. Graphical abstract summarizing the scheme of study.

Differential regulation of mRNAs and lncRNAs related to lipid metabolism in Duolang and Small Tail Han sheep.

The function of long non-coding RNA (lncRNA) can be achieved through the regulation of target genes, and the deposition of fat is regulated by lncRNA. Fat has an important effect on meat quality. However, there are relatively few studies on lncRNAs in the subcutaneous adipose tissue of Duolang sheep and Small Tail Han sheep. In this study, RNA-Seq technology and bioinformatics methods were used to identify and analyze the lncRNA and mRNA in the subcutaneous adipose tissue of the two breeds of sheep. The results showed that 107 lnRNAs and 1329 mRNAs were differentially expressed. The differentially expressed genes and lncRNA target genes were significantly enriched in the biosynthesis of unsaturated fatty acids signaling pathway, fatty acid metabolism, adipocyte differentiation and other processes related to fat deposition. Among them, LOC105616076, LOC114118103, LOC105607837, LOC101116622, and LOC105603235 target FADS1, SCD, ELOVL6, HSD17B12 and HACD2, respectively. They play a key regulatory role in the biosynthesis of unsaturated fatty acids. This study lays a foundation for the study of the molecular mechanism of lncRNA on fat development, and has reference value for studying the differences in fat deposition between Duolang sheep and Small Tail Han sheep.

Genome-Wide Analysis of microRNAs Identifies the Lipid Metabolism Pathway to Be a Defining Factor in Adipose Tissue From Different Sheep.

microRNAs are a class of important non-coding RNAs, which can participate in the regulation of biological processes. In recent years, miRNA has been widely studied not only in humans and mice, but also in animal husbandry. However, compared with other livestock and poultry breeds, the study of miRNA in subcutaneous adipose tissue of sheep is not comprehensive. Transcriptome analysis of miRNAs in subcutaneous adipose tissue of Duolang sheep, and Small Tail Han sheep was performed using RNA-Seq technology. Differentially expressed miRNAs were screened between different breeds. Target genes were predicted, and then the joint analysis of candidate genes were conducted based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, the RNA-Seq data were verified by real-time quantitative polymerase chain reaction (qRT-PCR). Herein, we identified 38 differentially expressed miRNAs (9 novel miRNAs and 29 known miRNAs). In addition, a total of 854 target genes were predicted by miRanda software. GO and KEGG pathway analysis demonstrated that regulation of lipolysis in adipocytes plays a key role in the deposition of subcutaneous adipose tissue in Duolang sheep and Small Tail Han sheep. The miRNAs might regulate fat deposits by regulating genes involved in regulation of lipolysis in adipocytes. Specifically, NC_ 040278.1_ 37602, oar-mir-493-3p, NC_ 040278.1_ 37521 and NC_ 040255.1_ 11627 might target PTGS2, AKT2, AKT3, and PIK3CA, respectively, and then play critical regulatory role. In conclusion, all the results provide a good idea for further revealing the mechanism of subcutaneous adipose tissue deposition and improving the meat production performance of sheep, and lay a foundation for promoting the development of animal husbandry.

The comprehensive detection of miRNA and circRNA in the regulation of intramuscular and subcutaneous adipose tissue of Laiwu pig.

circRNAs, as miRNA sponges, participate in many important biological processes. However, it remains unclear whether circRNAs can regulate lipid metabolism. This study aimed to explore the competing endogenouse RNA (ceRNA) regulatory network that affects the difference between intramuscular fat (IMF) and subcutaneous fat (SCF) deposition, and to screen key circRNAs and their regulatory genes. In this experiment, we identified 265 differentially expressed circRNAs, of which 187 up-regulated circRNA and 78 down-regulated circRNA in IMF. Subsequently, we annotated the function of DEcircRNA's host genes, and found that DEcircRNA's host genes were mainly involved in GO terms (including cellular response to fatty acids, lysophosphatidic acid acyltransferase activity, R-SMAD binding, etc.) and signaling pathways (fatty acid biosynthesis, Citrate cycle, TGF- ß Signal pathway) related to adipogenesis, differentiation and lipid metabolism. By constructing a circRNA-miRNA network, we screened out DEcircRNA that can competitively bind to more miRNAs as key circRNAs (circRNA_06424 and circRNA_08840). Through the functional annotation of indirect target genes and protein network analysis, we found that circRNA_06424 affects the expression of PPARD, MMP9, UBA7 and other indirect target genes by competitively binding to miRNAs such as ssc-miR-339-5p, ssc-miR-744 and ssc-miR-328, and participates in PPAR signaling pathway, Wnt signaling pathway, unsaturated fatty acid and other signaling pathways, resulting in the difference of fat deposition between IMF and SCF. This study provide a theoretical basis for further research investigating the differences of lipid metabolism in different adipose tissues, providing potential therapeutic targets for ectopic fat deposition and lipid metabolism diseases.

Genome-Wide Expression Profiling and Networking Reveals an Imperative Role of IMF-Associated Novel CircRNAs as ceRNA in Pigs.

Intramuscular fat (IMF) deposition is a biological process that has a strong impact on the nutritional and sensorial properties of meat, with relevant consequences on human health. Pork loins determine the effects of marbling on the sensory attributes and meat quality properties, which differ among various pig breeds. This study explores the crosstalk of non-coding RNAs with mRNAs and analyzes the potential pathogenic role of IMF-associated competing endogenous RNA (ceRNA) in IMF tissues, which offer a framework for the functional validation of key/potential genes. A high-throughput whole-genome transcriptome analysis of IMF tissues from longissimus dorsi muscles of Large White (D_JN) and Laiwu (L_JN) pigs resulted in the identification of 283 differentially expressed circRNAs (DECs), including two key circRNAs (circRNA-23437, circRNA-08840) with potential binding sites for multiple miRNAs regulating the whole network. The potential ceRNA mechanism identified the DEC target miRNAs-mRNAs involved in lipid metabolism, fat deposition, meat quality, and metabolic syndrome via the circRNA-miRNA-mRNA network, concluding that ssc-mir-370 is the most important target miRNA shared by both key circRNAs. TGM2, SLC5A6, ECI1, FASN, PER1, SLC25A34, SOD1, and COL5A3 were identified as hub genes through an intensive protein-protein interaction (PPI) network analysis of target genes acquired from the ceRNA regulatory network. Functional enrichments, pathway examinations, and qRT-PCR analyses infer their implications in fat/cholesterol metabolism, insulin secretion, and fatty acid biosynthesis. Here, circRNAs and miRNA sequencing accompanied by computational techniques were performed to analyze their expressions in IMF tissues from the longissimus dorsi muscles of two pig breeds. Their target gene evolutionary trajectories, expression profiling, functional enrichments, subcellular localizations, and structural advances with high-throughput protein modeling, following genomic organizations, will provide new insights into the underlying molecular mechanisms of adipocyte differentiation and IMF deposition and a much-needed qualitative framework for future research to improve meat quality and its role as a biomarker to treat lipid metabolic syndromes.

Identification and analysis of lncRNA, miRNA and mRNA related to subcutaneous and intramuscular fat in Laiwu pigs.

Background: Long non-coding RNAs (lncRNAs) regulate adipocyte differentiation and metabolism, However, their function on subcutaneous and intramuscular adipose tissues in pigs is unclear. Intramuscular fat (IMF) is an important indicator for evaluating meat quality. Breeds with high IMF content are often accompanied by high subcutaneous fat (SCF), which severely affects the meat rate of pigs. It is of great significance for porcine breeding to study the mechanism of lncRNA related to adipogenesis and lipid metabolism. Methods: We identified differentially expressed lncRNAs, miRNAs and mRNAs in subcutaneous and intramuscular adipose tissues in three female Laiwu pigs by deep RNA-sequencing(|log2foldchange|≥1, P_value ≤ 0.05). The gene expression profiles of IMF and SCF in Laiwu pigs were comparatively analyzed by Bioinformatics methods to identify key lncRNAs, miRNAs, and mRNAs associated with lipid metabolism and adipogenesis. Results: A total of 1209 lncRNAs (DElncRNAs), 286 miRNAs (DEmiRNAs), and 1597 mRNAs (DEgenes) were differentially expressed between two types of adipose. Among them, 17 DElncRNAs and 103 target genes play a role in the co-expression network, as well as 59 DElncRNAs, 44 DEmiRNAs, and 88 DEgenes involved in ceRNA network. In GO(Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of DElncRNAs their target genes involved in many adipogenesis and lipid metabolism biological processes and signaling pathways, such as PPAR signaling pathway, Wnt signaling pathway, MAPK signaling pathway. Conclusions: By constructing co-expression and ceRNAs network we found that Wnt signaling pathway play a critical regulatory role in intramuscular adipogenesis and lipid accumulation in Laiwu pigs. TCONS_00006525, TCONS_00046551 and TCONS_00000528 may target WNT5A, WNT10B and FDZ3 in co-expression network, TCONS_00026517 and other lncRNAs regulate the expression of PPARG, RXRG and SCD in ceRNA network, and were involved in Wnt signaling pathway. This study provides a theoretical basis for further understanding the post-transcriptional regulation mechanism of meat quality formation, predicting and treating diseases caused by ectopic fat.

Identification of potential miRNA-mRNA regulatory network and the key miRNAs in intramuscular and subcutaneous adipose.

Intramuscular fat (IMF) is an important indicator for evaluating meat quality. Breeds with high IMF content are often accompanied by high subcutaneous fat (SCF), severely affecting the meat rate of pigs. Studying the mechanisms of miRNAs related to lipogenesis and lipid metabolism has important implications for pig breeding. We constructed two small RNA libraries from intramuscular and subcutaneous fat to evaluate the patterns of lipogenesis in Laiwu pig, a Chinese breed. A total of 286 differentially expressed miRNAs (DEmiRNAs), including 193 known miRNA and 93 novel miRNAs, were identified from two types of adipose. GO and KEGG enrichment analysis for DEmiRNAs showed that their target genes involved in many adipogenesis and lipid metabolism biological processes and signaling pathways, such as Wnt signaling pathway,MAPK signaling pathway, Hippo signaling pathway, PI3K-Akt signaling pathway, Melanogenesis, Signaling pathways regulating pluripotency of stem cells and so on. Then, we constructed a miRNA-mRNA interaction network to find out which miRNAs were the key miRNAs of regulation in Wnt signaling pathway. In this pathway, miR-331-3p, miR-339-5p, miR-874 and novel346_mature target PPARD, WNT10B, RSPO3, WNT2B. This study provides a theoretical basis for further understanding the post-transcriptional regulation mechanism of meat quality formation and predicting and treating diseases caused by ectopic fat.

Genome-wide transcriptome profiling uncovers differential miRNAs and lncRNAs in ovaries of Hu sheep at different developmental stages.

Ovary development is an important determinant of the procreative capacity of female animals. Here, we performed genome-wide sequencing of long non-coding RNAs (lncRNAs) and mRNAs on ovaries of 1, 3 and 8 months old Hu sheep to assess their expression profiles and roles in ovarian development. We identified 37,309 lncRNAs, 45,404 messenger RNAs (mRNAs) and 330 novel micro RNAs (miRNAs) from the transcriptomic analysis. Six thousand, seven hundred and sixteen (6716) mRNAs and 1972 lncRNAs were significantly and differentially expressed in ovaries of 1 month and 3 months old Hu sheep (H1 vs H3). These mRNAs and target genes of lncRNAs were primarily enriched in the TGF-ß and PI3K-Akt signalling pathways which are closely associated with ovarian follicular development and steroid hormone biosynthesis regulation. We identified MSTRG.162061.1, MSTRG.222844.7, MSTRG.335777.1, MSTRG.334059.16, MSTRG.188947.6 and MSTRG.24344.3 as vital genes in ovary development by regulating CTNNB1, CCNA2, CDK2, CDC20, CDK1 and EGFR expressions. A total of 2903 mRNAs and 636 lncRNAs were differentially expressed in 3 and 8 months old ovaries of Hu sheep (H3 vs H8); and were predominantly enriched in PI3K-Akt, progesterone-mediated oocyte maturation, estrogen metabolism, ovulation from the ovarian follicle and oogenesis pathways. These lncRNAs were also found to regulate FGF7, PRLR, PTK2, AMH and INHBA expressions during follicular development. Our result indicates the identified genes participate in the development of the final stages of follicles and ovary development in Hu sheep.