Whole-Transcriptome Analysis Sheds Light on the Biological Contexts of Intramuscular Fat Deposition in Ningxiang Pigs.

The quality of pork is significantly impacted by intramuscular fat (IMF). However, the regulatory mechanism of IMF depositions remains unclear. We performed whole-transcriptome sequencing of the longissimus dorsi muscle (IMF) from the high (5.1 ± 0.08) and low (2.9 ± 0.51) IMF groups (%) to elucidate potential mechanisms. In summary, 285 differentially expressed genes (DEGs), 14 differentially expressed miRNAs (DEMIs), 83 differentially expressed lncRNAs (DELs), and 79 differentially expressed circRNAs (DECs) were identified. DEGs were widely associated with IMF deposition and liposome differentiation. Furthermore, competing endogenous RNA (ceRNA) regulatory networks were constructed through co-differential expression analyses, which included circRNA-miRNA-mRNA (containing 6 DEMIs, 6 DEGs, 47 DECs) and lncRNA-miRNA-mRNA (containing 6 DEMIs, 6 DEGs, 36 DELs) regulatory networks. The circRNAs sus-TRPM7_0005, sus-MTUS1_0004, the lncRNAs SMSTRG.4269.1, and MSTRG.7983.2 regulate the expression of six lipid metabolism-related target genes, including PLCB1, BAD, and GADD45G, through the binding sites of 2-4068, miR-7134-3p, and miR-190a. For instance, MSTRG.4269.1 regulates its targets PLCB1 and BAD via miRNA 2_4068. Meanwhile, sus-TRPM7_0005 controls its target LRP5 through ssc-miR-7134-3P. These findings indicate molecular regulatory networks that could potentially be applied for the marker-assisted selection of IMF to enhance pork quality.

Integrated analysis of muscle transcriptome, miRNA, and proteome of Chinese indigenous breed Ningxiang pig in three developmental stages.

The Ningxiang pig, a distinguished local breed in China, is recognized for its good meat quality traits. This study examines the proteomics of Ningxiang pigs at three developmental stages and delves into the upstream transcriptomics of these proteomics. Such an analysis facilitates a deeper understanding of the molecular interplay between proteins and transcriptomes in the Ningxiang pig muscle, influencing muscle growth and development. In this research, we analyzed the muscles of Ningxiang pigs at three developmental stages: 30 days in weaned piglets, 90 days in nursery pigs, and 210 days in late fattening pigs. There a total of 16 differentially co-expressed miRNAs (ssc-miRNA-1, ssc-miRNA-378, ssc-miRNA-143, ssc-miRNA-30e, etc.), 74 differentially co-expressed mRNA (PLIN3, CPT2, IGF2 and HSP90AB1, etc.) have been identified in the three stages. 572 differentially abundant proteins (DAPs) (APOC3, NDUFA2, HSPD1, ATP5E, PDHA1, etc.) were readily identified by comparing different time periods. According to the KEGG enrich pathway results that DAPs most enriched in growth and development pathways, immune mechanism pathways and maintaining functions of physical. Through short time-series expression miner (STEM) association analysis, a total of 571 negative miRNA-mRNA interaction pairs and 2 negative miRNA-mRNA-protein (Chr05_11955-Pig.17268.1-ATP5F1B, ssc-miR-194a-3p-Pig.15802.1-ACY1) interaction pairs were found. Our study provides a theoretical basis on molecular mechanism for the study of IMF deposition, muscle growth and immunity in Ningxiang pig breed.

Characterization and Function Analysis of miRNA Editing during Fat Deposition in Chinese Indigenous Ningxiang Pigs.

This study aimed to identify active miRNA editing sites during adipose development in Ningxiang pigs and analyze their characteristics and functions. Based on small RNA-seq data from the subcutaneous adipose tissues of Ningxiang pigs at four stages-30 days (piglet), 90 days (nursery), 150 days (early fattening), and 210 days (late fattening)-we constructed a developmental map of miRNA editing in the adipose tissues of Ningxiang pigs. A total of 505 miRNA editing sites were identified using the revised pipeline, with C-to-U editing types being the most prevalent, followed by U-to-C, A-to-G, and G-to-U. Importantly, these four types of miRNA editing exhibited base preferences. The number of editing sites showed obvious differences among age groups, with the highest occurrence of miRNA editing events observed at 90 days of age and the lowest at 150 days of age. A total of nine miRNA editing sites were identified in the miRNA seed region, with significant differences in editing levels (p < 0.05) located in ssc-miR-23a, ssc-miR-27a, ssc-miR-30b-5p, ssc-miR-15a, ssc-miR-497, ssc-miR-15b, and ssc-miR-425-5p, respectively. Target gene prediction and KEGG enrichment analyses indicated that the editing of miR-497 might potentially regulate fat deposition by inhibiting adipose synthesis via influencing target binding. These results provide new insights into the regulatory mechanism of pig fat deposition.

Comprehensive analysis of transcriptomic and metabolomic profiles uncovered the age-induced dynamic development pattern of subcutaneous fat in Ningxiang pig.

Enhancing meat production and quality is the eternal theme for pig breeding industries. Fat deposition has always been the focus of research in practical production because it is closely linked to pig production efficiency and pork quality. In the current study, multi-omics techniques were performed to explore the modulatory mechanisms of backfat (BF) accumulation at three core developmental stages for Ningxiang pigs. Our results identified that 15 differentially expressed genes (DEGs) and 9 significantly changed metabolites (SCMs) contributed to the BF development via the cAMP signaling pathway, regulation of lipolysis in adipocytes, and biosynthesis of unsaturated fatty acids. Herein, we found a series of candidate genes such as adrenoceptor beta 1 (ADRB1), adenylate cyclase 5 (ADCY5), ATPase Na+/K+ transporting subunit beta 1 (ATP1B1), ATPase plasma membrane Ca2+ transporting 3 (ATP2B3), ATPase Na+/K+ transporting subunit alpha 2 (ATP1A2), perilipin 1 (PLIN1), patatin like phospholipase domain containing 3 (PNPLA3), ELOVL fatty acid elongase 5 (ELOVL5) and metabolites like epinephrine, cAMP, arachidonic acid, oleic acid, linoleic acid, and docosahexaenoic acid existed age-specificeffects and played important roles in lipolysis, fat accumulation, and fatty acid composition. Our findings provide a reference for molecular mechanisms in BF tissue development and the optimization of carcass quality.

Integrated analysis of circRNA, lncRNA, miRNA and mRNA to reveal the ceRNA regulatory network of postnatal skeletal muscle development in Ningxiang pig.

Introduction: The development of skeletal muscle is regulated by regulatory factors of genes and non-coding RNAs (ncRNAs). Methods: The objective of this study was to understand the transformation of muscle fiber type in the longissimus dorsi muscle of male Ningxiang pigs at four different growth stages (30, 90, 150, and 210 days after birth, n = 3) by histological analysis and whole transcriptome sequencing. Additionally, the study investigated the expression patterns of various RNAs involved in muscle fiber transformation and constructed a regulatory network for competing endogenous RNA (ceRNA) that includes circular RNA (circRNA)/long non-coding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA). Results: Histomorphology analysis showed that the diameter of muscle fiber reached its maximum at 150 days after birth. The slow muscle fiber transformation showed a pattern of initial decrease followed by an increase. 29,963 circRNAs, 2,683 lncRNAs, 986 miRNAs and 22,411 mRNAs with expression level ≥0 were identified by whole transcriptome sequencing. Furthermore, 642 differentially expressed circRNAs (DEc), 505 differentially expressed lncRNAs (DEl), 316 differentially expressed miRNAs (DEmi) and 6,090 differentially expressed mRNAs (DEm) were identified by differential expression analysis. Functions of differentially expressed mRNA were identified by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). GO enrichment analysis indicates that 40 known genes and 6 new genes are associated with skeletal muscle development. Additionally, KEGG analysis shows that these genes regulate skeletal muscle development via MAPK, FoxO, Hedgehog, PI3K-Akt, Notch, VEGF and other signaling pathways. Through protein-protein interaction (PPI) and transcription factor prediction (TFP), the action mode of skeletal muscle-related genes was explored. PPI analysis showed that there were stable interactions among 19 proteins, meanwhile, TFP analysis predicted 22 transcription factors such as HMG20B, MYF6, MYOD1 and MYOG, and 12 of the 19 interacting proteins were transcription factors. The regulatory network of ceRNA related to skeletal muscle development was constructed based on the correlation of various RNA expression levels and the targeted binding characteristics with miRNA. The regulatory network included 31 DEms, 59 miRNAs, 667 circRNAs and 224 lncRNAs. conclusion: Overall, the study revealed the role of ceRNA regulatory network in the transformation of skeletal muscle fiber types in Ningxiang pigs, which contributes to the understanding of ceRNA regulatory network in Ningxiang pigs during the skeletal muscle development period.

Molecular Mechanism of MYL4 Regulation of Skeletal Muscle Development in Pigs.

The processes of muscle growth and development, including myoblast proliferation, migration, differentiation, and fusion, are modified by a variety of regulatory factors. MYL4 plays an important role in atrial development, atrial cardiomyopathy, muscle-fiber size, and muscle development. The structural variation (SV) of MYL4 was found via the de novo sequencing of Ningxiang pigs, and the existence of SV was verified in the experiments. The genotype distribution of Ningxiang pigs and Large White pigs was detected, and it was found that Ningxiang pigs were mainly of the BB genotype and that Large White pigs were mainly of the AB genotype. However, the molecular mechanisms behind the MYL4-mediated regulation of skeletal muscle development need to be deeply explored. Therefore, RT-qPCR, 3'RACE, CCK8, EdU, Western blot, immunofluorescence, flow cytometry, and bioinformation analysis were used to explore the function of MYL4 in myoblast development. The cDNA of MYL4 was successfully cloned from Ningxiang pigs, and its physicochemical properties were predicted. The expression profiles in six tissues and four stages of Ningxiang pigs and Large White pigs were found to be the highest in the lungs and 30 days after birth. The expression of MYL4 increased gradually with the extension of the myogenic differentiation time. The myoblast function test showed that the overexpression of MYL4 inhibited proliferation and promoted apoptosis and differentiation. The knockdown of MYL4 showed the opposite result. These results enhance our understanding of the molecular mechanisms of muscle development and provide a solid theoretical foundation for further exploring the role of the MYL4 gene in muscle development.

Circular Intronic RNA circTTN Inhibits Host Gene Transcription and Myogenesis by Recruiting PURB Proteins to form Heterotypic Complexes.

Muscle cell growth plays an important role in skeletal muscle development. Circular RNAs (circRNAs) have been proven to be involved in the regulation of skeletal muscle growth and development. In this study, we explored the effect of circTTN on myoblast growth and its possible molecular mechanism. Using C2C12 cells as a functional model, the authenticity of circTTN was confirmed by RNase R digestion and Sanger sequencing. Previous functional studies have showed that the overexpression of circTTN inhibits myoblast proliferation and differentiation. Mechanistically, circTTN recruits the PURB protein on the Titin (TTN) promoter to inhibit the expression of the TTN gene. Moreover, PURB inhibits myoblast proliferation and differentiation, which is consistent with circTTN function. In summary, our results indicate that circTTN inhibits the transcription and myogenesis of the host gene TTN by recruiting PURB proteins to form heterotypic complexes. This work may act as a reference for further research on the role of circRNA in skeletal muscle growth and development.

Transcriptome Analysis Reveals the Age-Related Developmental Dynamics Pattern of the Longissimus Dorsi Muscle in Ningxiang Pigs.

The growth and development of the Longissimus Dorsi muscle are complex, playing an important role in the determination of pork quality. The study of the Longissimus Dorsi muscle at the mRNA level is particularly crucial for finding molecular approaches to improving meat quality in pig breeding. The current study utilized transcriptome technology to explore the regulatory mechanisms of muscle growth and intramuscular fat (IMF) deposition in the Longissimus Dorsi muscle at three core developmental stages (natal stage on day 1, growing stage on day 60, and finishing stage on day 210) in Ningxiang pigs. Our results revealed 441 differentially expressed genes (DEGs) in common for day 1 vs. day 60 and day 60 vs. day 210, and GO (Gene Ontology) analysis showed that candidate genes RIPOR2, MEGF10, KLHL40, PLEC, TBX3, FBP2, and HOMER1 may be closely related to muscle growth and development, while KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that DEGs (UBC, SLC27A5, RXRG, PRKCQ, PRKAG2, PPARGC1A, PLIN5, PLIN4, IRS2, and CPT1B) involved the PPAR (Peroxisome Proliferator-Activated Receptor) signaling pathway and adipocytokine signaling pathway, which might play a pivotal role in the regulation of IMF deposition. PPI (Protein-Protein Interaction Networks) analysis found that the STAT1 gene was the top hub gene. Taken together, our results provide evidence for the molecular mechanisms of growth and development and IMF deposition in Longissimus Dorsi muscle to optimize carcass mass.

miR-100-5p Regulates Skeletal Muscle Myogenesis through the Trib2/mTOR/S6K Signaling Pathway.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial regulatory roles in many biological processes, including the growth and development of skeletal muscle. miRNA-100-5p is often associated with tumor cell proliferation and migration. This study aimed to uncover the regulatory mechanism of miRNA-100-5p in myogenesis. In our study, we found that the miRNA-100-5p expression level was significantly higher in muscle tissue than in other tissues in pigs. Functionally, this study shows that miR-100-5p overexpression significantly promotes the proliferation and inhibits the differentiation of C2C12 myoblasts, whereas miR-100-5p inhibition results in the opposite effects. Bioinformatic analysis predicted that Trib2 has potential binding sites for miR-100-5p at the 3'UTR region. A dual-luciferase assay, qRT-qPCR, and Western blot confirmed that Trib2 is a target gene of miR-100-5p. We further explored the function of Trib2 in myogenesis and found that Trib2 knockdown markedly facilitated proliferation but suppressed the differentiation of C2C12 myoblasts, which is contrary to the effects of miR-100-5p. In addition, co-transfection experiments demonstrated that Trib2 knockdown could attenuate the effects of miR-100-5p inhibition on C2C12 myoblasts differentiation. In terms of the molecular mechanism, miR-100-5p suppressed C2C12 myoblasts differentiation by inactivating the mTOR/S6K signaling pathway. Taken together, our study results indicate that miR-100-5p regulates skeletal muscle myogenesis through the Trib2/mTOR/S6K signaling pathway.

Genome-Wide Association Analysis Identifies Genomic Regions and Candidate Genes for Growth and Fatness Traits in Diannan Small-Ear (DSE) Pigs.

In the livestock industry, the growth and fatness traits are directly related to production efficiency and economic profits. As for Diannan small-ear (DSE) pigs, a unique indigenous breed, the genetic architecture of growth and fatness traits is still elusive. The aim of this study was to search the genetic loci and candidate genes associated with phenotypic traits in DSE pigs using GWAS based on the Geneseek Porcine 50K SNP Chip data. A total of 22,146 single nucleotide polymorphisms (SNPs) were detected in 265 DSE pigs and used for Genome-wide association studies (GWAS) analysis. Seven SNPs were found to be associated with back height, chest circumference, cannon bone circumference, and backfat thickness at the suggestive significance level. Based on gene annotation results, these seven SNPs were, respectively, mapped to the following candidate genes, VIPR2, SLC10A2, NUCKS1, MCT1, CHCHD3, SMOX, and GPR1, which are mainly involved with adipocyte differentiation, lipid metabolism, skeletal muscle development, and average daily weight gain. Our work offers novel insights into the genetic architecture of economically important traits in swine and may play an important role in breeding using molecular markers in the DSE breed.

Association of gut microbiota and SCFAs with finishing weight of Diannan small ear pigs.

Finishing weight is a key economic trait in the domestic pig industry. Evidence has linked the gut microbiota and SCFAs to health and production performance in pigs. Nevertheless, for Diannan small ear (DSE) pigs, a specific pig breed in China, the potential effect of gut microbiota and SCFAs on their finishing weight remains unclear. Herein, based on the data of the 16S ribosomal RNA gene and metagenomic sequencing analysis, we found that 13 OTUs could be potential biomarkers and 19 microbial species were associated with finishing weight. Among these, carbohydrate-decomposing bacteria of the families Streptococcaceae, Lactobacillaceae, and Prevotellaceae were positively related to finishing weight, whereas the microbial taxa associated with intestinal inflammation and damage exhibited opposite effects. In addition, interactions of these microbial species were found to be linked with finishing weight for the first time. Gut microbial functional annotation analysis indicated that CAZymes, such as glucosidase and glucanase could significantly affect finishing weight, given their roles in increasing nutrient absorption efficiency. Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthologies (KOs) and KEGG pathways analysis indicated that glycolysis/gluconeogenesis, phosphotransferase system (PTS), secondary bile acid biosynthesis, ABC transporters, sulfur metabolism, and one carbon pool by folate could act as key factors in regulating finishing weight. Additionally, SCFA levels, especially acetate and butyrate, had pivotal impacts on finishing weight. Finishing weight-associated species Prevotella sp. RS2, Ruminococcus sp. AF31-14BH and Lactobacillus pontis showed positive associations with butyrate concentration, and Paraprevotella xylaniphila and Bacteroides sp. OF04-15BH were positively related to acetate level. Taken together, our study provides essential knowledge for manipulating gut microbiomes to improve finishing weight. The underlying mechanisms of how gut microbiome and SCFAs modulate pigs' finishing weight required further elucidation.

miR-708-5p Regulates Myoblast Proliferation and Differentiation.

MicroRNAs (miRNAs) are key regulators involved in the myogenic process in skeletal muscles. miR-708-5p plays an important role in various biochemical and physiological processes, but its function in skeletal myogenesis remain unclear. In this study, we first explored the effects of miR-708-5p on C2C12 proliferation and differentiation by overexpression and interference experiments. Then, we predicted the target genes of miR-708-5p and analyzed their function. We found that miR-708-5p was gradually increased during myoblast differentiation. Overexpression of miR-708-5p significantly inhibited cell proliferation and promoted the differentiation of myoblasts. A total of 253 target genes were predicted using a bioinformatics approach. These genes were significantly enriched in muscle growth-related GO terms and KEGG pathways, such as actin filament organization, actin cytoskeleton organization, PI3K-Akt pathway, insulin pathway, and Jak-STAT pathway. Among them, Pik3ca, Pik3r3, and Irs1 were considered to be the key target genes of miR-708-5p. To sum up, miR-708-5p inhibited C2C12 cells proliferation and promoted C2C12 cells differentiation. Its target genes significantly enriched in GO terms and KEGG pathways related to the development and growth of muscle. Our results provided a basis for studies on the function and mechanism of miR-708-5p regulating skeletal muscle growth and development.

Effects of age on subcutaneous adipose tissue proteins in Chinese indigenous Ningxiang pig by TMT-labeled quantitative proteomics.

Adipose tissue not only affects meat quality and animal productivity, but also participates in inflammation and immunity. Ningxiang pig is famous for their excellent meat quality, disease resistance and tolerance of roughage. It is not yet well known how proteins in adipose tissue is dynamically regulated during the growth of Ningxiang pig. This report studies the proteomic changes in subcutaneous adipose tissue in Ningxiang pigs to gain a better understanding of the molecular mechanism of fat development during the growth period. By TMT-labeled quantitative proteomic analysis of subcutaneous adipose tissue of 9 purebred Ningxiang pigs of different ages, we identified 2533 unique proteins and 716 differentially abundant proteins (DAPs), of which more than half of the DAPs were concentrated in the 90d-210d period. Retrograde endocannabinoid signaling was only significantly enriched in DAPs of N90d vs N30d, Alcoholism and Graft-versus-host disease were only significantly enriched in DAPs of N210d vs N90d. Proteins related to dilated cardiomyopathy was found to be an important pathway in fat development and lipid metabolism. A variety of novel DAPs involved in maintaining mitochondrial function and cell viability, such as NDUFS6, SDHB, COX5A, ATP5D and TNNT1, which play a role in controlling the prediction networks, may indirectly regulate the development and functional maintenance of adipocytes. SIGNIFICANCE: These age-dependent DAPs discovered in this study may help expand the understanding of the molecular mechanisms of the development, function maintenance and transformation of adipose tissue in Ningxiang pig for developing new strategies for improving meat quality and pig breeding in the future.

Spatiotemporal Regulation of Circular RNA Expression during Liver Development of Chinese Indigenous Ningxiang Pigs.

BACKGROUND: There have been many studies on the relationship between circRNAs and fat deposition. Although the liver is a central organ for fat metabolism, there are few reports on the relationship between circRNAs in the liver and fat deposition. METHODS: In this study, we systematically analyzed circular RNAs in the liver of Ningxiang pigs, at four time points after birth (30 days, 90 days, 150 days and 210 days). RESULTS: A total of 3705 circRNAs were coexpressed in four time periods were found, and KEGG analysis showed that the significantly upregulated pathways were mainly enriched in lipid metabolism and amino acid metabolism, while significantly downregulated pathways were mainly related to signal transduction, such as ECM-receptor interaction, MAPK signaling pathway, etc. Short time-series expression miner (STEM) analysis showed multiple model spectra that were significantly enriched over time in the liver. By constructing a competing endogenous RNA (ceRNA) regulatory network, 9187 pairs of networks related to the change in development time were screened. CONCLUSIONS: The expression profiles of circRNAs in Ningxiang pig liver were revealed at different development periods, and it was determined that there is differential coexpression. Through enrichment analysis of these circRNAs, it was revealed that host genes were involved in metabolism-related signaling pathways and fatty acid anabolism. Through STEM analysis, many circRNAs involved in fat metabolism, transport, and deposition pathways were screened, and the first circRNA-miRNA-mRNA regulation network map in Ningxiang pig liver was constructed. The highly expressed circRNAs related to fat deposition were verified and were consistent with RNA-Seq results.

Preliminary identification and analysis of differential RNA editing between higher and lower backfat thickness pigs using DNA-seq and RNA-seq data.

RNA editing is an essential post-transcriptional regulatory mechanism. However, few studies have investigated the functional RNA edits in the economic traits of livestock on a genome-wide scale. Pigs are one of the most important livestock species and their fat is the principal organ involved in the regulation of adipose deposition. Here, we used three full-sibling pairs, with each pair comprising a pig with higher backfat (BF) thickness and lower backfat thickness, to identify RNA editing events based on whole-genome and transcriptome sequencing data. A total of 60,903 non-redundant RNA editing sites with 59,472 (97.7%) A-to-G edits were detected using a revised bioinformatics pipeline. A specific sequence context with G preference was found one base downstream of the edited site, and the editing level was associated with the distribution of nucleotides across nearly sites. Moreover, the A-to-G editing sites mostly occurred in the pig-special short interspersed nuclear elements, Pre0_SS. Comparing the difference between pigs with higher BF and lower BF, we found 211 differentially edited sites (DESites). Functional enrichment analyses revealed a significant enrichment of genes containing DESites in terms of adipose deposition. The DESites located in the six adipose-related genes (SKP1, GSK3B, COL5A3, MDM4, NT5C2, and DENND2A) were selected as candidate RNA editing sites associated with adipose deposition, and thus require further evaluation. This study mined the potentially functional RNA editing sites in pig adipose tissue and indicated that RNA editing may play an important role in adipose deposition, which provides a new insight into the post-transcriptionally mediated regulation mechanism of fat development.

miR-875 Serves as A Candidate Biomarker for Detection and Prognosis and Is Correlated with PHH3 Index Levels in Breast Cancer Patients.

BACKGROUND: miRNAs play crucial roles in cancers. This study investigated the potential value of miR-875 to be a detective or/and prognostic marker and evaluate the correlation between its expression and PHH3 index levels in breast cancer. METHODS: miR-875 expression was determined in breast cancer serum and tissues by a quantitative real-time reverse transcription-polymerase chain reaction. The detective value of serum miR-875 expression for breast cancer was assessed by receiver operating characteristic analysis. Then the associations of miR-875 expression with clinical characteristics of patients and overall survival were evaluated by χ2 test and Kaplan-Meier curve methods, respectively. RESULTS: The expression of miR-875 was increased in both breast cancer serum and tissues compared with respective controls. The high miR-875 expression in serum and tissues was associated with positive lymph node metastasis and advanced TNM stages. Besides, miR-875 expression in tissues was positively associated with the PHH3 index. And serum miR-875 could screen breast cancer patients from healthy individuals. Moreover, breast cancer patients with both high expression of miR-875 and PHH3 index had shorter overall survival. CONCLUSION: This study suggested that miR-875 expression may be suitable as a potential breast cancer detection and prognosis biomarker. And the miR-875 expression in tissues was positively associated with the PHH3 index, suggesting that miR-875 expression may be involved in tumor proliferation of breast cancer.

Long-read assembly of the Chinese indigenous Ningxiang pig genome and identification of genetic variations in fat metabolism among different breeds.

Advances in long-read sequencing technology and genome assembly provide an opportunity to improve the pig genome and reveal the full range of structural variations (SVs) between local Chinese and European pigs. To date, little is known about the genomes of some unique Chinese indigenous breeds, such as the Ningxiang pig. Here, we report the sequencing and assembly of a highly contiguous Ningxiang pig genome (NX) via an integration of PacBio single-molecule real-time sequencing, Illumina next-generation sequencing, BioNano optical mapping and Hi-C (chromosome conformation capture) approaches. The assembled genome comprises 2.44 Gb with a contig N50 of 26.1 Mb and 418 contigs in total. These contigs are organized into 121 scaffolds with a scaffold N50 of 139.0 Mb. More than 99.1% of the assembled sequence could be localized to 19 pseudochromosomes and is annotated with 20,914 protein-coding genes and 34.04% repetitive sequences. Comparisons between the NX and European Duroc assemblies revealed many SVs in genes involved in the immune system, nervous system, lipid metabolism and environmental adaptation. The genetic variants include 47 Chinese domestic pig-specific SVs and the associated 74 genes may contribute to the differences in domestic traits compared to European pigs. Moreover, single nucleotide polymorphisms (SNPs) identified from whole genome resequencing data of 73 Chinese pigs, representing 17 geographically isolated breeds, showed their specific genetic variations, population structure and evolutionary patterns. Finally, we explore transcriptional regulation in the first intron of the MYL4 gene, as the genomic SV (281-bp deletion) in Ningxiang pig promotes its subcutaneous fat compared to European pig breeds. This work identifies a set of Asian-specific SVs and SNPs, which will be important resources for modern pig breeding and genetic conservation.

Integrated Analysis of Liver Transcriptome, miRNA, and Proteome of Chinese Indigenous Breed Ningxiang Pig in Three Developmental Stages Uncovers Significant miRNA-mRNA-Protein Networks in Lipid Metabolism.

Liver is an important metabolic organ of mammals. During each transitional period of life, liver metabolism is programmed by a complex molecular regulatory system for multiple physiological functions, many pathways of which are regulated by hormones and cytokines, nuclear receptors, and transcription factors. To gain a comprehensive and unbiased molecular understanding of liver growth and development in Ningxiang pigs, we analyzed the mRNA, microRNA (miRNA), and proteomes of the livers of Ningxiang pigs during lactation, nursery, and fattening periods. A total of 22,411 genes (19,653 known mRNAs and 2758 novel mRNAs), 1122 miRNAs (384 known miRNAs and 738 novel miRNAs), and 1123 unique proteins with medium and high abundance were identified by high-throughput sequencing and mass spectrometry. We show that the differences in transcriptional, post-transcriptional, or protein levels were readily identified by comparing different time periods, providing evidence that functional changes that may occur during liver development are widespread. In addition, we found many overlapping differentially expressed genes (DEGs)/differentially expressed miRNAs (DEMs)/differentially expressed proteins (DEPs) related to glycolipid metabolism in any group comparison. These overlapping DEGs/DEMs/DGPs may play an important role in functional transformation during liver development. Short Time-series Expression Miner (STEM) analysis revealed multiple expression patterns of mRNA, miRNA, and protein in the liver. Furthermore, several diverse key Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including immune defense, glycolipid metabolism, protein transport and uptake, and cell proliferation and development, were identified by combined analysis of DEGs and DGPs. A number of predicted miRNA-mRNA-protein pairs were found and validated by qRT-PCR and parallel reaction monitoring (PRM) assays. The results provide new and important information about the genetic breeding of Ningxiang pigs, which represents a foundation for further understanding the molecular regulatory mechanisms of dynamic development of liver tissue, functional transformation, and lipid metabolism.

Spatiotemporal Regulation and Functional Analysis of Circular RNAs in Skeletal Muscle and Subcutaneous Fat during Pig Growth.

Recently, thousands of circular RNAs have been reported in different pig breeds. However, researches on the temporal and spatial expression patterns of circRNA over the period of animal growth are limited. Here, we systematically analyzed circRNAs in skeletal muscle and subcutaneous fat in four growth time points (30 days, 90 days, 150 days and 210 days after birth) of a Chinese native pig breed, Ningxiang pigs. A total of 1171 differentially expressed (DE) circRNAs between muscle and fat were identified, including 562 upregulated and 609 downregulated circRNAs. KEGG pathway enrichment analysis of these DE circRNAs revealed that host genes were mainly involved in glycogen metabolism signaling pathways, muscle development signaling pathways such as ErbB pathway and adipocytokine signaling pathways and AMPK signaling pathways and fatty acid biosynthesis. The circRNAs have striking spatiotemporal specificity in the form of dynamic expression at 90 d. Short Time-Series Expression Miner analysis showed multiple model spectra that are significantly enriched with time changes in muscle and fat. Our findings provide new ideas and perspectives about the role of circular RNAs and their targeting relations with mRNA and miRNA in skeletal muscle and fat tissue during pig growth.

Integrated Analysis of lncRNA and mRNA in Subcutaneous Adipose Tissue of Ningxiang Pig.

Ningxiang pigs, a Chinese bred pig known for its tender meat and high quality unsaturated fatty acids. This study discovers the transcription profiles and functional networks in long non-coding RNA (lncRNA) and messenger RNA (mRNA) in subcutaneous adipose tissue. Subcutaneous adipose tissue was collected from piglet, nursery pig, early fattening, and late fattening stage of Ningxiang piglets, and lncRNA and mRNA transcription of each stage was profiled. A total of 339,204,926 (piglet), 315,609,246 (nursery), 266,798,202 (early fattening), and 343,740,308 (late fattening) clean reads were generated, and 2872 novel lncRNAs were identified. Additionally, 10,084 differential mRNAs (DEmRNAs) and 931 differential lncRNAs were determined. Most DEmRNAs were up-regulated in the piglet stage, while they were down-regulated in late fattening stage. A complicated interaction between mRNAs and lncRNAs was identified via STEM and WGCNA, demonstrated that lncRNAs are a significant regulatory component in mRNAs. The findings showed that modules 2 and 5 have a similar mode of transcription for both mRNA and lncRNA, and were mainly participated in steroid biosynthesis, glycosphingolipid biosynthesis, metabolic pathways, and glycerolipid metabolism. The mRNAs and lncRNAs transcription levels of both modules was higher in the early and late fattening stage, which may be due to the active activity of the metabolism in relation to fatty acids, sterols, steroids, and lipids in the subcutaneous adipose tissue during the early and late fattening stage. These findings could be expected to result in further research of the functional properties of lncRNA from subcutaneous adipose tissue at different stages of development in Ningxiang pigs.