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RNA-seq analysis of mammary tissue reveals persistent changes in gene expression profiles dependent upon the level of folic acid intake in C56BL/6 mice.

ABSTRACT: Purpose: To identify, using RNA-sequencing, differentially expressed genes in the mammary gland of mice supplemented with FA. We aimed to ascertain whether gene expression is persistently altered after increased FA supplementation, whether such alterations are related to genes implicated in epigenetic regulation and disease risk. Methods: Mammary gland mRNA profiles of mice 4 weeks after folic acid supplementation (5mg/kg vs 1mg/kg) during adulthood were generated by deep sequencing using RNA sequencing. The sequence reads that passed quality filters were aligned to the mouse genome using TopHat and analyzed at the gene level using DESeq2. qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped reads to the mouse genome (build mm10) and identified 222 genes differentially expressed between the two group (adjsuted p value <0.05). 191 of these were upregulated and 31 were downregulated. Geneset enrichment analysis identified several enriched pathways, including epithelial mesenchymal transition and myogenesis. Conclusions: Our study provides novel insights into the persistent effects of FA intake on the C57BL/6 mammary transcriptome and identifies putative gene targets and molecular pathways that help to understand how increasing FA intake may modulate the long term expression of gene networks and disease pathways.

ORGANISM(S): Mus musculus

PROVIDER: GSE153732 | GEO | 2020/09/15

REPOSITORIES: GEO

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Project description:The objective of this study was to determine the effect of the DGAT1 K232A polymorphism on the global mRNA expression pattern of genes in the mammary gland tissue of grazing dairy cows in order to get more insight into the effects of this polymorphism on the physiology of the mammary glandgland of grazing dairy cows. Microarray analysis was used to identify genes whose expression was affected by the DGAT1 polymorphism in the mammary gland biopsies of 9 A232A cows, 13 K232A cows, and 4 K232K cows. The Microarray Analysis of Variance (MAANOVA) and Factor Analysis for Multiple Testing method (FAMT) were used to associate the expression of the genes present on Affymettrix Bovine Genome Arrays with the DGAT1 polymorphism. The data was also analysed at the level of functional modules by gene set enrichment analysis. In this experimental setting, DGAT1 polymorphism did not modify milk yield and composition significantly, although changes occurred in the yields of C14:0, C16:1cis-9, and some long chain fatty acids in milk. The DGAT1 polymorphism resulted in 30 differentially expressed genes related to cell growth, proliferation and development, signalling, remodelling and immune system. At the functional level, the pathways most affected by DGAT1 polymorphism were related to lipid biosynthesis, which likely reflected counter mechanisms of mammary tissue to respond to changes in milk FA composition, signalling, as well as immune system responses. A total of 28 Holstein-Friesian dairy cows in mid-lactation (DIM; 153 M-BM-1 32.8 days), milk yield (25.7 M-BM-1 3.08 kg/d) and fat content (4.3 M-BM-1 0.12%) were used in the study. Two consecutive milk samples (a.m. and p.m. milking) were obtained and pooled. One aliquot was stored at 4M-BM-0C until analysis of fat, protein and lactose percentage, and another aliquot was frozen at -20M-BM-0C until analysis for FA composition by gas chromatography. Specific details regarding the analysis of FA in milk are presented in Mach et al. (2011). Approximately 750 to 1,000 mg of mammary tissue from each cow was obtained by surgical biopsy. One part of the tissue was used for isolation of DNA and the other for extraction of the RNA. Genotyping of the DGAT1 polymorphism was performed using a TaqMan allelic discrimination method in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) as described by Schennink et al. (2007). The genotype at the DGAT1 locus was designated KK, KA, or AA for homozygous Lysine, heterozygous Lysine/Alanine, or homozygous Alanine, respectively. Microarray analysis was used to identify genes whose expression was affected by the DGAT1 polymorphism in the mammary gland biopsies of 11 A232A cows, 13 K232A cows, and 4 K232K cows. Total RNA from mammary gland tissue (50-100 mg) was isolated using TRIzol reagent (Invitrogen, Breda, The Netherlands), following the manufacturerM-bM-^@M-^Ys instructions. The RNA purity and concentrations were determined using a NanoDrop ND-1000 spectrophotometer (Isogen, Maarssen, the Netherlands), and the RNA quality was assessed using the BioAnalyzer 2100 (Agilent Technologies, Amsterdam, the Netherlands). The RNA of each biopsy was amplified, biotin-labeled, and hybridized to single-dye Affymetrix GeneChipM-BM-. Bovine Genome Array (#900493) by ServiceXS (Leiden, the Netherlands)

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RNA-seq analysis of mammary tissue reveals persistent changes in gene expression profiles dependent upon the level of folic acid intake in C56BL/6 mice.

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