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Identification of Molecular Mechanisms Related to Pig Fatness at the Transcriptome and miRNAome Levels.

ABSTRACT: Fat deposition and growth rate are closely related to pork quality and fattening efficiency. The next-generation sequencing (NGS) approach for transcriptome and miRNAome massive parallel sequencing of adipocyte tissue was applied to search for a molecular network related to fat deposition in pigs. Pigs were represented by three breeds (Large White, Pietrain, and Hampshire) that varied in fat content within each breed. The obtained results allowed for the detection of significant enrichment of Gene Ontology (GO) terms and pathways associated directly and indirectly with fat deposition via regulation of fatty acid metabolism, fat cell differentiation, inflammatory response, and extracellular matrix (ECM) organization and disassembly. Moreover, the results showed that adipocyte tissue content strongly affected the expression of leptin and other genes related to a response to excessive feed intake. The findings indicated that modification of genes and miRNAs involved in ECM rearrangements can be essential during fat tissue growth and development in pigs. The identified molecular network within genes and miRNAs that were deregulated depending on the subcutaneous fat level are proposed as candidate factors determining adipogenesis, fatness, and selected fattening characteristics in pigs.

SUBMITTER: Ropka-Molik K

PROVIDER: S-EPMC7348756 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Identification of Molecular Mechanisms Related to Pig Fatness at the Transcriptome and miRNAome Levels.

Ropka-Molik Katarzyna K Pawlina-Tyszko Klaudia K Żukowski Kacper K Tyra Mirosław M Derebecka Natalia N Wesoły Joanna J Szmatoła Tomasz T Piórkowska Katarzyna K

Genes 20200529 6

Fat deposition and growth rate are closely related to pork quality and fattening efficiency. The next-generation sequencing (NGS) approach for transcriptome and miRNAome massive parallel sequencing of adipocyte tissue was applied to search for a molecular network related to fat deposition in pigs. Pigs were represented by three breeds (Large White, Pietrain, and Hampshire) that varied in fat content within each breed. The obtained results allowed for the detection of significant enrichment of Ge ...[more]

PMID: 32485856

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Project description:Purpose: The aim of present research was to identify miRNAs potentially related with fattness traits in pigs Methods: miRNA-seq analysis was performed on subcutaneous fat samples collected from 22 pigs representing two sire-line breeds – Pietrain and Hampshire and one dam-line – Large White. Based on dissection data, pigs were selected from a larger population in terms of fatness traits to obtain the most extreme groups in each breed. The cDNA libraries were constructed form 300 ng of total RNA with the use NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, Ipswich, MA, United States) according to the protocol. The quantification of obtained libraries was performed on Qubit 2.0 (Invitrogen, Life Technologies) and TapeStation 2200 (D1000 ScreenTape; Agilent). The RNA-seq was performed in 36 single-end cycles on HiScanSQ platform (Illumina) with the use of ruSeq SR Cluster Kit v3- CBOT-HS and TruSeq SBS Kit v 3 - HS (Illumina). Each library was sequences in 4 technical replications. The DEGs were detected using DESEq2 software and validation was performed by qPCR. Results: RNA-seq approach allowed to identify miRNAs differentially expressed between pigs with various fatness traits in each analyzed breeds: 64 for Pietrain pigs; 41 for Hampshire and 46 for Large White pigs (pvalue <0.05; fold change≤1.5). The comparison of obtained miRNAs sets showed 13 miRNAs identified in all three breeds. The Gene Ontology analysis (mirPath v.3.0 DIANA Tools web with DIANA—TarBase v7.0 as a reference) confirmed that detected miRNAs were involved in fatty acid biosynthesis (pvalue <1e-325); fatty acid metabolism (pvalue 6.661338e-16); ECM-receptor interaction (pvalue <1e-325). The most important targeted genes regulated by identified miRNAs were: fatty acid synthase – FASN; Acetyl-CoA carboxylase 1 – ACACA; Malonyl CoA-acyl carrier protein transacylase – MCAT and acetyl-CoA acyltransferase 1 and 2 (ACAA1 and ACAA2 genes). Conclusions: Obtained results allow to propose the panel of miRNAs, which can be related with fatness in pig. Such analysis can be the basis of future research in terms of identification of molecular mechanisms related with adipogenesis and fatness traits in pigs.

Project description:Iberian ham production includes both purebred (IB) and Duroc-crossbred (IBxDU) Iberian pigs, which show important differences in meat quality and production traits, such as muscle growth and fatness. This experiment was conducted to investigate gene expression differences, transcriptional regulation and genetic polymorphisms that could be associated with the observed phenotypic differences between IB and IBxDU pigs. Nine IB and 10 IBxDU pigs were slaughtered at birth. Morphometric measures and blood samples were obtained and samples from Biceps femoris muscle were employed for compositional and transcriptome analysis by RNA-Seq technology. Phenotypic differences were evident at this early age, including greater body size and weight in IBxDU and greater Biceps femoris intramuscular fat and plasma cholesterol content in IB newborns. We detected 149 differentially expressed genes between IB and IBxDU neonates (p < 0.01 and Fold-Change > 1. 5). Several were related to adipose and muscle tissues development (DLK1, FGF21 or UBC). The functional interpretation of the transcriptomic differences revealed enrichment of functions and pathways related to lipid metabolism in IB and to cellular and muscle growth in IBxDU pigs. Protein catabolism, cholesterol biosynthesis and immune system were functions enriched in both genotypes. We identified transcription factors potentially affecting the observed gene expression differences. Some of them have known functions on adipogenesis (CEBPA, EGRs), lipid metabolism (PPARGC1B) and myogenesis (FOXOs, MEF2D, MYOD1), which suggest a key role in the meat quality differences existing between IB and IBxDU hams. We also identified several polymorphisms showing differential segregation between IB and IBxDU pigs. Among them, non-synonymous variants were detected in several transcription factors as PPARGC1B and TRIM63 genes, which could be associated to altered gene function. Taken together, these results provide information about candidate genes, metabolic pathways and genetic polymorphisms potentially involved in phenotypic differences between IB and IBxDU pigs associated to meat quality and production traits.

Project description:This work was aimed at evaluating loin transcriptome and metabolic pathway differences between the two main Serbian local pig breeds with divergent characteristics regarding muscle growth and fatness, as well as exploring nutrigenomic effects of tannin supplementation in Mangalitsa (MA) pigs. The study comprised 24 Mangalitsa and 10 Moravka (MO) males, which were kept under identical management conditions. Mangalitsa animals were divided in two nutritional groups (n = 12) receiving a standard (control) or tannin-supplemented diet (1.5%; MAT). Moravka pigs were fed the standard mixture. All animals were slaughtered at a similar age; 120 kg of average live weight (LW) and loin tissue was used for RNA-seq analysis. Results showed 306 differentially expressed genes (DEGs) according to breed, enriched in genes involved in growth, lipid metabolism, protein metabolism and muscle development, such as PDK4, FABP4, MYOD1 and STAT3, as well as a relevant number of genes involved in mitochondrial respiratory activity (MT-NDs, NDUFAs among others). Oxidative phosphorylation was the most significantly affected pathway, activated in Mangalitsa muscle, revealing the basis of a different muscle metabolism. Also, many other relevant pathways were affected by breed and involved in oxidative stress response, fat accumulation and development of skeletal muscle. Results also allowed the identification of potential regulators and causal networks such as those controlled by FLCN, PPARGC1A or PRKAB1 with relevant regulatory roles on DEGs involved in mitochondrial and lipid metabolism, or IL3 and TRAF2 potentially controlling DEGs involved in muscle development. The Tannin effect on transcriptome was small, with only 23 DEGs, but included interesting ones involved in lipid deposition such as PPARGC1B. The results indicate a significant effect of the breed on muscle tissue gene expression, affecting relevant biological pathways and allowing the identification of strong regulatory candidate genes to underlie the gene expression and phenotypic differences between the compared groups.

Project description:Transcriptome profiles established using high-throughput sequencing can be effectively used for screening genome-wide differentially expressed genes (DEGs). RNA sequences (from RNA-seq) and microRNA sequences (from miRNA-seq) from the tissues of longissimus dorsi muscle of two indigenous Chinese pig breeds (Diannan Small-ear pig [DSP] and Tibetan pig [TP]) and two introduced pig breeds (Landrace [LL] and Yorkshire [YY]) were examined using HiSeq 2000 to identify and compare the differential expression of functional genes related to muscle growth and lipid deposition. We obtained 27.18 G clean data through the RNA-seq and detected that 18,208 genes were positively expressed and 14,633 of them were co-expressed in the muscle tissues of the four samples. In all, 315 DEGs were found between the Chinese pig group and the introduced pig group, 240 of which were enriched with functional annotations from the David database and significantly enriched in 27 Gene Ontology (GO) terms that were mainly associated with muscle fiber contraction, cadmium ion binding, response to organic substance and contractile fiber part. Based on functional annotation, we identified 85 DEGs related to growth traits that were mainly involved in muscle tissue development, muscle system process, regulation of cell development, and growth factor binding, and 27 DEGs related to lipid deposition that were mainly involved in lipid metabolic process and fatty acid biosynthetic process. With miRNA-seq, we obtained 23.78 M reads and 320 positively expressed miRNAs from muscle tissues, including 271 known pig miRNAs and 49 novel miRNAs. In those 271 known miRNAs, 20 were higher and 10 lower expressed in DSP-TP than in LL-YY. The target genes of the 30 miRNAs were mainly participated in MAPK, GnRH, insulin and Calcium signaling pathway and others involved cell development, growth and proliferation, etc. Combining the DEGs and the differentially expressed (DE) miRNAs, we drafted a network of 46 genes and 18 miRNAs for regulating muscle growth and a network of 15 genes and 16 miRNAs for regulating lipid deposition. We identified that CAV2, MYOZ2, FRZB, miR-29b, miR-122, miR-145-5p and miR-let-7c, etc, were key genes or miRNAs regulating muscle growth, and FASN, SCD, ADORA1, miR-4332, miR-182, miR-92b-3p, miR-let-7a and miR-let-7e, etc, were key genes or miRNAs regulating lipid deposition. The quantitative expressions of eight DEGs and seven DE miRNAs measured with real-time PCR certified that the results of differential expression genes or miRNAs were reliable. Thus, 18,208 genes and 320 miRNAs were positively expressed in porcine longissimus dorsi muscle. We obtained 85 genes and 18 miRNAs related to muscle growth and 27 genes and 16 miRNAs related to lipid deposition, which provided new insights into molecular mechanism of the economical traits in pig.

Dataset Information

Identification of Molecular Mechanisms Related to Pig Fatness at the Transcriptome and miRNAome Levels.

Publications

Identification of Molecular Mechanisms Related to Pig Fatness at the Transcriptome and miRNAome Levels.

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