RNA-Seq of wild type and miR-574 knockout mouse hearts at baseline

Purpose: The target mRNAs of miR-574 in mouse heart are unknown. The goal of this study is to use next generation sequencing (NGS) RNA-seq followed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods to identify the downstream target genes that are directly regulated by miR-574 in murine hearts at the mRNA level. Results: Using a well established data analysis workflow from the Genomic Research Center from University of Rochester Medical Center, we analyzed RNA-seq data. We have identified novel genes that are regulated by miR-574 via miRNA-induced silencing complex (miRISC). We defined differentially regulated genes (either upregulated or downregulated) with statistical significance in total RNA expression change (p value <0.05 for differential expressed genes). We identified 34 upregulated genes involved in the small molecule metabolism and the mitochondrial function, and 49 downregulated genes in the electron transport chain (ETC) complex, oxidative phosphorylation, and tricarboxylic acid cycle. Conclusions: Among 34 upregulated genes, eight genes contain the predicted target seed sequence of miR-574-5p and Fam210a (family with sequence similarity 210 member A) is the only upregulated gene that contains the target seed sequence of both the guide strand miR-574-5p and the passenger strand miR-574-3p. We confirmed that FAM210A was the downstream target of both miR-574-5p and miR-574-3p in vivo and in vitro. Based on our results, we conclude that miR-574 downregulates a limited number of mitochondrial function related genes including FAM210A in mouse hearts at baseline. Overall design: We extracted totoal RNA from three pairs of fresh P60 heart tissues from WT and miR-574 KO mice (under C57BL6/J genetic background) at baseline. The total RNA samples were subjected to next generation RNA deep sequencing (RNA-Seq). All the extracted RNA samples were treated with DNase I to remove potential genomic DNA followed by purification using phenol:chloroform:isoamyl alcohol for RNA-Seq. Raw reads were generated from the Illumina HiSeq2500 sequencer in the Genomic Core from Case Western Reserve University.