Transcriptome-wide analysis of RNA m6A methylation regulation of muscle development in Queshan Black pigs.

BACKGROUND: N6-methyladenosine (m6A) refers to the methylation modification of N6 position of RNA adenine, a dynamic reversible RNA epigenetic modification that plays an important regulatory role in a variety of life processes. In this study, we used MeRIP-Seq and RNA-Seq of the longissimus dorsi (LD) muscle of adult (QA) and newborn (QN) Queshan Black pigs to screen key genes with m6A modification involved in muscle growth by bioinformatics analysis. RESULTS: A total of 23,445 and 25,465 m6A peaks were found in the whole genomes of QA and QN, respectively. Among them, 613 methylation peaks were significantly different (DMPs) and 579 genes were defined as differentially methylated genes (DMGs). Compared with the QN group, there were 1,874 significantly differentially expressed genes (DEGs) in QA group, including 620 up-regulated and 1,254 down-regulated genes. In order to investigate the relationship between m6A and mRNA expression in the muscle of Queshan Black pigs at different periods, a combined analysis of MeRIP-Seq and RNA-Seq showed that 88 genes were significantly different at both levels. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes results showed that DEGs and DMGs were mainly involved in skeletal muscle tissue development, FoxO signaling pathway, MAPK signaling pathway, insulin signaling pathway, PI3K-Akt signaling pathway, and Wnt signaling pathway. Four DEGs (IGF1R, CCND2, MYOD1 and FOS) and four DMGs (CCND2, PHKB, BIN1 and FUT2), which are closely related to skeletal muscle development, were selected as candidate genes for verification, and the results were consistent with the sequencing results, which indicated the reliability of the sequencing results. CONCLUSIONS: These results lay the foundation for understanding the specific regulatory mechanisms of growth in Queshan Black pigs, and provide theoretical references for further research on the role of m6A in muscle development and breed optimization selection.

Effect of miR-149-5p on intramuscular fat deposition in pigs based on metabolomics and transcriptomics.

As one of the important traits in pig production, meat quality has important research significance and value. Intramuscular fat (IMF) content is one of the most important factors affecting pork quality. Many experimental studies have shown that IMF content is closely related to the flavor, tenderness, and juiciness of pork. Therefore, it is of great significance to study the mechanism of porcine IMF deposition. Previous research indicated that miR-149-5p promoted the proliferation of porcine intramuscular (IM) preadipocytes and decreased their ability to differentiate, albeit the exact mechanism of action is unknown. In vitro, foreign pigs showed increased miR-149-5p expression and reduced fat deposition when compared to Queshan Black pigs. This study conducted metabolomics and transcriptomics analyses of porcine IM preadipocytes overexpressing miR-149-5p to verify their effects on lipid formation. According to metabolomics analysis, the overexpression of miR-149-5p has significantly altered the lipid, organic acid, and organic oxygen metabolites of porcine IM preadipocytes. Specially speaking, it has changed 115 metabolites, including 105 up-regulated and 10 down-regulated ones, as well as the composition of lipid, organic acid, and organic oxygen metabolism-related metabolites. RNA-seq analysis showed that overexpression of miR-149-5p significantly altered 857 genes, of which 442 were up-regulated, and 415 were down-regulated, with enrichment to MAPK, IL-17, PI3K-Akt, and ErbB signaling pathways. We found that overexpression of miR-149-5p inhibited adipogenic differentiation by changing cAMP signaling pathway in porcine IM preadipocytes. In addition, the overexpression of miR-149-5p may affect the transport of Cu2+ by targeting ATP7A and inhibiting adipogenic differentiation. These findings elucidate the regulatory function of miR-149-5p in porcine IM preadipocytes, which may be a key target for controlling pork quality.

Proteomic analysis of boar sperm with differential ability of liquid preservation at 17 °C.

Understanding the liquid preservation ability of boar sperm is pivotal for efficient management and breeding of livestock. Although sperm proteins play an important role in semen quality and freezability, how the levels of protein change in boar sperm with different liquid preservation abilities at 17 °C remains unclear. In this study, two groups of boar sperm with extreme difference in liquid preservation ability, namely the good preservation ability (GPA) and the poor preservation ability (PPA) groups, were selected by evaluating sperm motility parameters on the 7th day of liquid preservation at 17 °C. Quantitative proteomics based on tandem mass tag (TMT) labeling was used, sperm proteomic characteristics from two groups were analyzed, and potentially key proteins related to the fluid preservation ability of sperm were identified. A total of 187 differentially expressed proteins (DEPs) were identified among 2791 quantified proteins, including 85 upregulated, and 102 downregulated proteins. Further, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the DEPs revealed that they were enriched in GO terms associated with response to oxidative stress, enzyme activity related to oxidative stress or redox reactions, and several metabolic activities. The significant KEGG pathways included peroxisome, metabolic pathways, selenocompound metabolism, and collection duct acid secretion. In addition, analysis of protein-protein interactions further identified 8 proteins that could be used as biomarker candidates, including GPX5, GLRX, ENO4, QPCT, BBS7, OXSR1, DHRS4 and AP2S1, which may play an essential role in indicating the liquid preservation ability of boar sperm. These findings in this study provide new insights into the underlying molecular mechanisms of the liquid preservation ability of boar sperm. Moreover, the selected candidate proteins can serve as a reference for evaluating sperm quality or preservation ability in boars and their application in related biotechnologies.

Expression Profile and Regulatory Properties of m6A-Modified circRNAs in the Longissimus Dorsi of Queshan Black and Large White Pigs.

It is well known that N6-methyladenosine (m6A) is the most abundant modification in linear RNA molecules, but many circRNA molecules have now been found to have a wide range of m6A modification sites as well. However, there are few relevant studies and information on the expression profile and functional regulatory properties of m6A-modified circRNAs (m6A-circRNAs) in longissimus dorsi. In this study, a total of 12 putative m6A-circRNAs were identified and characterized in the longissimus dorsi of Queshan Black and Large White pigs-8 of them were significantly more expressed in the longissimus dorsi of Queshan Black than in Large White pigs, while the other 4 were the opposite. These 12 putative m6A-circRNAs were also found to act as miRNA sponge molecules to regulate fat deposition by constructing the ceRNA regulatory network. Enrichment analysis also revealed that the 12 m6A-circRNAs parent genes and their adsorbed miRNA target genes were widely involved in fat deposition and cell proliferation and differentiation-related pathways, such as the HIF-1 signaling pathway, the pentose phosphate pathway, the MAPK signaling pathway, the glycosphingolipid biosynthesis-lacto and neolacto series, and the TNF signaling pathway, suggesting that the analyzed m6A-circRNAs may be largely involved in the formation of pork quality. These results provide new information to study the regulatory properties of m6A-circRNAs in the formation of pork quality.

CircGUCY2C regulates cofilin 1 by sponging miR-425-3p to promote the proliferation of porcine skeletal muscle satellite cells.

Circular ribonucleic acids (or circRNAs) are an emerging class of endogenous noncoding RNAs that are involved in physiological and pathological processes. Increasing evidence suggests that circRNAs play an important regulatory role in skeletal muscle development and meat quality regulation. In this study, it was found that circGUCY2C exhibits a high expression level in the longissimus dorsi muscle. It shows resistance to RNase R and additionally promotes the mRNA expression of cyclin-dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA). Specifically, it was observed that the overexpression of circGUCY2C could promote the transition of porcine skeletal muscle satellite cells into the S and G2 phases of the cell cycle and that it regulates the proliferation of porcine skeletal muscle satellite cells. In contrast, miR-425-3p plays the opposite role and has an inhibitory effect on the proliferation of porcine skeletal muscle satellite cells. MiR-425-3p has been described as a target of circGUCY2C; consequently, the depletion of miR-425-3p promoted the proliferation of porcine skeletal muscle satellite cells. CFL1 (cofilin 1) is a target of miR-425-3p, and circGUCY2C upregulated CFL1 expression by inhibiting miR-425-3p. Collectively, our research outcomes demonstrate that circGUCY2C significantly influences the proliferation of porcine skeletal muscle satellite cells by selectively targeting the miR-425-3p-CFL1 axis, and our work partially clarified the role of circGUCY2C in porcine skeletal muscle satellite cells. Thus, the study provides new insight into the function of circGUCY2C and adds to the knowledge of the post-transcriptional regulation of pork quality.

Identification and Functional Prediction of Long Non-Coding RNA in Longissimus Dorsi Muscle of Queshan Black and Large White Pigs.

Long non-coding RNA (lncRNA) participates in the regulation of various biological processes, but its function and characteristics in intramuscular fat (IMF) deposition in different breeds of pigs have not been fully understood. IMF content is one of the important factors affecting pork quality. In the present study, the differentially expressed lncRNAs (DE lncRNAs) and their target genes were screened by comparing Queshan Black (QS) and Large White (LW) pigs based on RNA-seq. The results displayed 55 DE lncRNAs between QS and LW, 29 upregulated and 26 downregulated, with 172 co-located target genes, and 6203 co-expressed target genes. The results of GO and KEGG analysis showed that the target genes of DE lncRNAs were involved in multiple pathways related to lipogenesis and lipid metabolism, such as the lipid biosynthetic process, protein phosphorylation, activation of MAPK activity, and the Jak-STAT signaling pathway. By constructing regulatory networks, lincRNA-ZFP42-ACTC1, lincRNA-AMY2-STAT1, and/or lincRNA-AMY2/miR-204/STAT1 were sieved, and the results indicate that lncRNA could participate in IMF deposition through direct regulation or ceRNA. These findings provide a basis for analyzing the molecular mechanism of IMF deposition in pigs and lay a foundation for developing and utilizing high-quality resources of local pig breeds.

Genome-Wide Analysis of MAMSTR Transcription Factor-Binding Sites via ChIP-Seq in Porcine Skeletal Muscle Fibroblasts.

Myocyte enhancer factor-2-activating motif and SAP domain-containing transcriptional regulator (MAMSTR) regulates its downstream through binding in its promoter regions. However, its molecular mechanism, particularly the DNA-binding sites, and coregulatory genes are quite unexplored. Therefore, to identify the genome-wide binding sites of the MAMSTR transcription factors and their coregulatory genes, chromatin immunoprecipitation sequencing was carried out. The results showed that MAMSTR was associated with 1506 peaks, which were annotated as 962 different genes. Most of these genes were involved in transcriptional regulation, metabolic pathways, and cell development and differentiation, such as AMPK signaling pathway, TGF-beta signaling pathway, transcription coactivator activity, transcription coactivator binding, adipocytokine signaling pathway, fat digestion and absorption, skeletal muscle fiber development, and skeletal muscle cell differentiation. Lastly, the expression levels and transcriptional activities of PID1, VTI1B, PRKAG1, ACSS2, and SLC28A3 were screened and verified via functional markers and analysis. Overall, this study has increased our understanding of the regulatory mechanism of MAMSTR during skeletal muscle fibroblast development and provided a reference for analyzing muscle development mechanisms.

Seminal plasma metabolomics analysis of differences in liquid preservation ability of boar sperm.

The preservation of semen is pivotal in animal reproduction to ensure successful fertilization and genetic improvement of livestock and poultry. However, investigating the underlying causes of differences in sperm liquid preservation ability and identifying relevant biomarkers remains a challenge. This study utilized liquid chromatography-mass spectrometry (LC-MS) to analyze the metabolite composition of seminal plasma (SP) from two groups with extreme differences in sperm liquid preservation ability. The two groups namely the good liquid preservation ability (GPA) and the poor preservation ability (PPA). The aim was to explore the relationship between metabolite composition in SP and sperm liquid preservation ability, and to identify candidate biomarkers associated with this ability of sperm. The results revealed the identification of 756 metabolites and 70 differentially expressed metabolites (DEM) in the SP from two groups of boar semen with differing liquid preservation abilities at 17 °C. The majority of identified metabolites in the SP belonged to organic acids and derivatives as well as lipids and lipid-like molecules. The DEM in the SP primarily consisted of amino acids, peptides, and analogs. The Kyoto Encyclopedia of Genes and Genomes analysis also demonstrated that the DEM are mainly concentrated in amino acid synthesis and metabolism-related pathways (P < 0.05). Furthermore, eleven key metabolites were identified and six target amino acids were verified, and the results were consistent with the non-targeted metabolic analysis. These findings indicated that amino acids and their associated pathways play a potential role in determining boar sperm quality and liquid preservation ability. D-proline, arginine, L-citrulline, phenylalanine, leucine, DL-proline, DL-serine, and indole may serve as potential biomarkers for early assessment of boar sperm liquid preservation ability. The findings of this study are helpful in understanding the causes and mechanisms of differences in the liquid preservation ability of boar sperm, and provide valuable insights for improving semen quality assessment methods and developing novel extenders or protocols.

The current main method for preserving boar semen used in artificial insemination is liquid preservation. However, the preservation ability of boar sperm has large individual differences, and understanding the factors that influence this ability of sperm and identifying relevant biomarkers present challenges. Given the crucial role of seminal plasma (SP) in sperm survival and functionality maintenance, this study utilized liquid chromatography-mass spectrometry to analyze the metabolite composition of the SP from two groups with extreme differences in sperm liquid preservation ability. The aim was to explore the relationship between metabolites in SP and sperm liquid preservation ability, and identify candidate biomarkers associated with this ability of sperm. The results revealed the important role of amino acids and related pathways in determining boar sperm quality and storage ability. Several potential biomarkers for early evaluation of boar sperm liquid preservation ability, including D-proline, arginine, L-citrulline, phenylalanine, leucine, DL-proline, DL-serine, and indole were identified. This study provides valuable insights into the reasons and mechanisms behind differences in sperm liquid preservation ability and offers possibilities for improving semen preservation techniques.

Identification of Estrus in Sows Based on Salivary Proteomics.

The estrus cycle of multiparous Large White sows was divided into three stages to solve the problems of heavy workload and low accuracy of the traditional estrus identification method in pig production. Saliva protein was extracted from the oral saliva of multiparous sows. Label-free quantitative proteomics was used to detect salivary proteome, and MaxQuant software was used for quality control. Results showed that 246 proteins were identified in the three stages, where 40 proteins were significantly different (p < 0.05). The total proteins identified were enriched by STEM software and the protein function was annotated by using the ClueGO plug-in in the Cytoscape software. The results were enriched to eight different trends. The annotated items were related to protein synthesis and processing and estrogen response. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differential proteins involved in the pathways and entries included oocyte meiosis, response to estradiol, and oogenesis. Further interaction analysis showed that an interaction occurred between P00355, F1SHL9, P28491, F1SDR7, F2Z558, F1RYY6, and F2Z5G3 proteins. The findings served as a basis for revealing the changes in salivary protein content in the sow estrus cycle and provided a reference for the development of an estrus identification kit/test strip in the next step.

CircSETBP1 Acts as a MiR-149-5p Sponge to Promote Intramuscular Fat Deposition by Regulating CRTCs.

Circular RNAs (circRNAs) appear to be crucial in the process of adipogenesis according to mounting data. CircSETBP1 is a newly discovered circRNA associated with adipogenesis. Sequencing verification and RNase R treatment have confirmed the circular nature of circSETBP1 in porcine tissue. The precise function and mechanism of circSETBP1 in adipocyte biology are still unclear. Cell counting kit-8 (CCK8), Oil red O staining, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed in this investigation to reveal the functions of circSETBP1 and miR-149-5p in the growth and development of porcine intramuscular (IM) preadipocytes. CircSETBP1 overexpression accelerated cell differentiation while reducing cell proliferation. The opposite outcome was produced by overexpressing miR-149-5p. Meanwhile, circSETBP1 down-regulated the expression of miR-149-5p and miR-149-5p restrained the expression of CRTC1/CRTC2. CircSETBP1 was directly targeted by miR-149-5p, and CRTC1/CRTC2 were the target genes of miR-149-5p using bioinformatic analysis, the dual-Luciferase reporter system, and qRT-PCR. In conclusion, circSETBP1 controls the proliferation and differentiation of porcine IM preadipocytes and 3T3-L1 cells by regulating the miR-149-5p/CRTCs axis. The results of this study not only illuminate the molecular mechanism of circSETBP1/miR-149-5p involved in the deposition of porcine intramuscular fat (IMF), but they also provide a significant theoretical reference for raising quality of pork.

Construction and Comprehensive Analysis of miRNAs and Target mRNAs in Longissimus dorsi Muscle of Queshan Black and Large White Pigs.

A miRNA-mRNA combination analysis was performed on the longissimus dorsi muscle of adult Queshan Black and Large White pigs by RNA-seq technology to reveal the molecular mechanism affecting pork quality traits. The sequencing results showed that 39 miRNAs were differentially expressed between Queshan Black and Large White pigs, which targeted 5234 mRNAs, and 15 differentially expressed miRNAs targeted 86 differentially expressed mRNAs. The qRT-PCR results showed that miRNAs showed similar expression patterns to RNA-seq. The GO analysis indicated that differentially expressed miRNAs with differential target mRNAs were primarily involved in biological processes such as phospholipase activity, MAP-kinase scaffold activity, lipase activity, and regulation of the extent of cell growth. The KEGG analysis also revealed that such mRNAs were significantly enriched in the ECM-receptor interaction, sphingolipid metabolism, apoptosis, PI3K-Akt signaling pathway, and AMPK signaling pathway. In addition, software predictions showed that 17 (13 of which were upregulated and four were downregulated) of 39 differentially expressed miRNAs targeted 118 negatively correlated expression mRNAs. The upregulated miRNAs contained 103 negatively correlated target mRNAs, whereas the downregulated miRNAs contained 15 negatively correlated target mRNAs. The GO analysis showed that such mRNAs were primarily involved in MAP-kinase scaffold activity, myoblast development, and peptidyl-lysine methylation, and the KEGG analysis showed significant enrichment in ECM-receptor interaction and focal adhesion. The functional enrichment analysis of miRNA target genes revealed that miR-328 was screened out as a key miRNA, and preliminary functional validation was performed. Moreover, the overexpressed miR-328 could affect the expression of proliferation-related genes, such as CDK2, CDK4, CCNB1, CCND1, CCNE1, and PCNA. These results indicated that miR-328 may regulate fat deposition and affect meat quality by influencing related pathways. This study revealed that the miRNA-mRNA regulatory axis affects fat deposition and skeletal muscle development, which provides a theoretical basis for further study on the molecular mechanism of meat quality.