GSE122816
Identification of molecular mechanisms related with fatness in pig at transcriptome and miRNAome level
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. Overall design: The fat (subcutaneous fat tissue) microRNA sequencing was performed for 22 samples collected form pigs representing three breeds – Large White, Pietrain and Hampshire, using Illumina HiScan SQ  in 36 single-end cycles.