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RNA sequencing identifies differentially expressed genes in adult left ventricles following in utero caffeine exposure during embryonic day 6.5-9.5

ABSTRACT: Purpose: This study aims to identify the differentially expressed genes in adult left ventricles treated with caffeine during early embryogenesis. Methods: Pregnant CD-1 mice were treated with vehicle (normal saline) or 20 mg/kg caffeine via i.p. injection once daily during embryonic day (E) 6.5-9.5. Pups were born and raised to one year of age. Left ventricles from adult male mice were dissected and used for total RNA isolation with the RNeasy Plus Mini kit (Valencia, CA, USA). mRNA was isolated from total RNA using NEXTflex™ Poly(A) Beads (Bioo Scientific, Austin, TX, USA). Sequencing libraries were prepared with the NEBNext® mRNA Library Prep Master Mix Set for Illumina (NEB, Ipswich, MA, USA) and the NEBNext Multiplex Oligos for Illumina (NEB). Illumina-adapted libraries (n=3/treatment) were pooled at equal molar ratio and sequenced with one High Output 1x75 cycles run on a NextSeq500 sequencer (Illumina, San Diego, CA, USA). The fastq files generated from RNA-Seq were uploaded to the UF Research Computing Galaxy instance developed by the University of Florida. The data were cleaned with the following steps: removed sequencing artifacts, trimmed 5’ or 3’ ends with low scores, removed adaptor contamination, and filtered low quality reads by the FastQC program. The remaining high quality RNA-Seq reads were mapped to the mouse genome (mm10) with the Tophat2 tool. Counting of RNA-Seq reads were performed with HTSeq. Differential expression (DE) of genes between treatments was analyzed using R package EdgeR, with Ensembl Mus_GRCm38.79.gtf as the reference annotation. Genes with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Results: DE analysis revealed that 85 genes were up-regulated, and 31 genes were down-regulated in the adult left ventricles treated with in utero caffeine.

ORGANISM(S): Mus musculus

PROVIDER: GSE79013 | GEO | 2017/02/09

SECONDARY ACCESSION(S): PRJNA314693

REPOSITORIES: GEO

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Project description:Purpose: This study aimed to identify differentially expressed genes including alternative splice variants in embryonic ventricles following in utero caffeine treatment. Methods: Pregnant CD-1 mice were injected with 20 mg/kg of caffeine or vehicle control daily from embryonic day (E) 6.5-9.5. On E10.5, total RNA was isolated from embryonic ventricles and used for transcriptomic RNA sequencing with Illumina HiSeq 2000 (1X75bp). RNA-seq reads were aligned to the mouse genome (build mm10) with the Tophat for Illumina tool in the PSU galaxy platform. Counting and annotation of RNA-seq reads as well as alternative splicing analysis were performed with Partek Genomics Suite version 6.11. Differential expression of gene and transcript reads between treatments was analyzed with R package EdgeR. Genes/transcripts with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Differentially expressed genes were defined as genes with altered expression at either gene or transcript level. Unique differentially expressed genes were identified by combining the results from annotations with the RefSeq Transcripts (2013-05-10) or Ensembl Transcripts release 71 databases. Results: Differential expression analysis revealed that 59 genes and 451 transcripts were significantly up-regulated, and 65 genes and 398 transcripts were down-regulated by prenatal caffeine treatment (fold change >1.5 or <-1.5; p-value with FDR<0.05). In total, 900 unique genes were identified to have altered expression either at the gene or transcription level. Further analysis with Partek GS revealed that 183 genes had abnormal alternative splicing at the exon level after in utero caffeine treatment. Conclusions: In utero caffeine exposure caused gene expression changes in embryonic ventricles and these changes may lead to long-term effects on cardiac morphology and function. mRNA profiles in E10.5 heart ventricles treated with caffeine were generated by deep sequencing (n=2 for vehicle, n=3 for caffeine), using Illumina HiSeq 2000.

Project description:Our previous research demonstrated that caffeine exposure on embryonic day (E) 8.5 increased body weight, altered cardiac morphology and function in adult male mice. However, these adverse effects were not observed in adenosine A1 receptor knockout (A1AR-/-) mice. Our hypothesis is that A1AR action mediates changes in DNA methylation patterns in mice exposed in utero to caffeine and that these changes in methylation lead to long-term effects in adult mice. To test this hypothesis, DNA Methylation 2.1M Deluxe Promoter Arrays (NimbleGen) were used to examine the methylation patterns of DNA isolated from adult left ventricles of the A1AR knockout line exposed to 20 mg/kg caffeine at E8.5 in utero. In A1AR+/+ mice, 4,896 hypermethylated and 2,823 hypomethylated regions were discovered in the left ventricles of the caffeine group compared to the normal saline group. In A1AR-/- mice, 1,024 hypermethylated and 1,757 hypomethylated regions were found in the caffeine group. The differentially methylated regions (DMRs) in the caffeine treated A1AR+/+ hearts mapped to 6,148 genes, 4,853 promoters, 4,111 primary transcripts, 816 CpG islands, and 98 miRNAs. Functional annotation clustering of genes revealed that many genes were involved in the development of hypertrophic cardiomyopathy and other heart diseases, which may explain our earlier findings of thickening of the left ventricular wall after in utero caffeine treatment. The methylation changes in several DMRs, mef2c, ins2, tnnt2, and myh6, were validated by bisulfite sequencing. In summary, in utero caffeine exposure caused DNA methylation changes in adult left ventricles and that these changes may be mediated by A1ARs. Pregnant mice were injected at embryonic day 8.5 with 0.9% NaCl (vehicle) or 20 mg/kg caffeine in vehicle i.p. Pups were born and raised until 8-10 weeks of age. Analysis was performed on the following groups vehicle A1AR+/+ (veh+/+), vehicle A1AR-/- (veh-/-), caffeine A1AR+/+ (caff+/+), and caffeine A1AR-/- (caff-/-). Genomic DNA from left ventricles of adult male mice was used. Two biological replicates were measured for each group.

Project description:Purpose: This study aims to identify the differentially expressed genes in embryonic cardiomyocytes following the knockdown of DNMT1 expression. Methods: Primary cardiomyocytes were isolated from mouse embryonic day (E) 13.5 ventricles, and cultured for 48 h at 37°C to reach 70-80% confluency. Cells were transfected with either Qiagen FlexiTube GeneSolution (with 4 siRNAs) DNMT1 siRNA (#GS13433) or AllStars negative control siRNA (#1027281) at 12 nM using lipofectamine RNAiMAX (n=3 per treatment). At 72 h after transfection, cells were released with Accutase (Innovative Cell Technologies, San Diego, CA) and dissociated with Accumax (Innovative Cell Technologies). To sort cardiomyocytes, dissociated cells were stained with anti-VCAM1 antibody conjugated with allophycocyanin (APC; BioLegend, San Diego, CA) and magnetically sorted using anti-APC microbeads and magnetic assisted cell sorting (MACS) columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Total RNA was isolated from the sorted cardiomyocytes with the RNAqueous®-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA). RNA-Seq libraries were prepared with the NEBNext® mRNA Library Prep Master Mix Set for Illumina and NEBNext Multiplex Oligos for Illumina (NEB, Ipswich, MA). The Illumina-adapted libraries were pooled at equal molar ratio and sequenced with one High Output 1x75 cycles run on a NextSeq500 sequencer (Illumina). The fastq files generated from RNA-Seq were uploaded to the UF Research Computing Galaxy instance developed by the University of Florida. The data were cleaned with the following steps: removed sequencing artifacts, trimmed 5’ or 3’ ends with low scores, removed adaptor contamination, and filtered low quality reads by the FastQC program. The remaining high quality RNA-Seq reads were mapped to the mouse genome (mm10) with the Tophat2 tool. Counting of RNA-seq reads were performed with HTSeq. Differential expression (DE) of genes between treatments was analyzed using two methods: R packages EdgeR and DEseq2, with Ensembl Mus_GRCm38.79.gtf as the reference annotation. Genes with false discovery rate (FDR) less than 0.05 and absolute fold change greater than 1.5 were considered as significant. Unique DE genes were identified by combining the results generated from the two analytical methods. Results: DE analysis by DEseq2 and EdgeR software revealed that 801 genes were up-regulated, and 494 genes were down-regulated after knockdown of DNMT1 expression for 72 h.

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RNA sequencing identifies differentially expressed genes in adult left ventricles following in utero caffeine exposure during embryonic day 6.5-9.5

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