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Identification of genes related with carcass fatness in pigs based on RNA-seq results

ABSTRACT: Purpose: RNA-seq method was applied to select genes expressed in fat tissue and pinpointed genes potentially associated with fatness traits in pigs. Methods: The RNA-seq analysis was performed on fat tissue collected from 16 Pulawska pigs (8 pigs per two groups - with high and low backfat thickness) maintained at the same environmental and feeding condition. The total RNA was isolated using by TriReagent and 300ng was used to cDNA libraries preparation. The NGS sequencing was performed on HiScan SQ (Illumina) in 90 single-end cycles. The quantifying transcript abundances was made using the RSEM supported by STAR aligner. The raw reads were aligned to the Sus scrofa reference genome. Differentially expressed genes were detected by DESeq2. The RNA-seq results were validated using by qPCR. Results: In the group of pigs characterized by thicker subcutaneous fat, 166 genes with increased expression were identified, including genes involved in biological processes associated with negative regulation of long-chain fatty acid transport (GO: 0010748) (IRS2, THBS1, AKT2), regulation of metabolic lipids ( GO: 0019216) (EEF1A2, IRS2, ACACA, SIK1, ADGRF5, IDH1, FGF2, PDE3B, FASN, AKT2, CYR61, CHD9, ME1, SCD), cellular response to hormonal stimuli (GO: 0032870) (IRS2, ACACA, SIK1 , GHR, NR4A1, PDE3B, PTGER2, AKT2, AACS, ADCY6, KAT2B, RHOQ, FOS). Genes involved in the signaling pathway responsible for the activation of gene expression by SREBF (R-SSC-2426168) (ACACA, FASN, CHD9, SCD ) and the insulin signaling pathway also were identified (RHOQ, SOCS3, IRS2, AKT2, PDE3B, PYGM, FASN, ACACA). Conclusions: The presented results allowed to pinpointed the interesting, candidate genes related with fat content in carcasses in pigs, which can be analyzed in future in the term of using in breeding practice as genetic markers. The study was financed from funds of the project -BIOSTRATEG, the decision number BIOSTRATEG2/297267/14/NCBR/2016.

ORGANISM(S): Sus scrofa

PROVIDER: GSE122349 | GEO | 2018/11/12

REPOSITORIES: GEO

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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:High-protein diets are known to reduce adiposity in the context of high carbohydrate and Western diets. However, few studies have investigated the specific high-protein effect on lipogenesis induced by a high-sucrose (HS) diet or fat deposition induced by high-fat feeding. We aimed to determine the effects of high protein intake on the development of fat deposition and partitioning in response to high-fat and/or HS feeding. A total of thirty adult male Wistar rats were assigned to one of the six dietary regimens with low and high protein, sucrose and fat contents for 5 weeks. Body weight (BW) and food intake were measured weekly. Oral glucose tolerance tests and meal tolerance tests were performed after 4th and 5th weeks of the regimen, respectively. At the end of the study, the rats were killed 2 h after ingestion of a calibrated meal. Blood, tissues and organs were collected for analysis of circulating metabolites and hormones, body composition and mRNA expression in the liver and adipose tissues. No changes were observed in cumulative energy intake and BW gain after 5 weeks of dietary treatment. However, high-protein diets reduced by 20 % the adiposity gain induced by HS and high-sucrose high-fat (HS-HF) diets. Gene expression and transcriptomic analysis suggested that high protein intake reduced liver capacity for lipogenesis by reducing mRNA expressions of fatty acid synthase (fasn), acetyl-CoA carboxylase a and b (Acaca and Acacb) and sterol regulatory element binding transcription factor 1c (Srebf-1c). Moreover, ketogenesis, as indicated by plasma β-hydroxybutyrate levels, was higher in HS-HF-fed mice that were also fed high protein levels. Taken together, these results suggest that high-protein diets may reduce adiposity by inhibiting lipogenesis and stimulating ketogenesis in the liver. Adult male Wistar rats were fed diets with varying amounts of protein, carbohydrates and fat for 5 weeks. At the end of the experiment, rats were killed and tanscriptome analysis was performed on pooled liver samples.

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Identification of genes related with carcass fatness in pigs based on RNA-seq results

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