Analysis of Wild Type and miR-21-/- Smooth Muscle Cell Transcriptomes after atherosclerosis

Purpose: The goals of this study are to investigate the effect of miR-21 knockout in aortic smooth muscle cell transcriptome profiling (RNA-seq) to understand how miR-21 regulates SMC plasticity during atherosclerosis. Methods: Aortic smooth muscle cell (SMC) mRNA profiles of 30-week-old wild-type (WT) and SMC specific miR-21 knockout (miR-21fl/flmTmG/Myh11ERT2) mice fed on western diet for 20 weeks were generated by deep sequencing, in triplicate. The sequence reads that passed quality filters were analyzed at the transcript isoform level . qRT–PCR validation was performed using TaqMan and SYBR Green assays to confirm miR-21 knockout. Results: Using an optimized data analysis workflow, we mapped about 60 million sequence reads per sample to the mouse genome (build mm10) and identified 22,836 transcripts in the SMCs of WT and miR-21fl/flmTmG/Myh11ERT2 mice. RNA-seq data revealed ~300 genes differentially expressed in miR-21 knockout SMCs after atherosclerosis. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized pathways that may contribute to SMC function during atherosclerosis progression. Conclusions: Our study represents the first detailed analysis of SMC lacking of miR-21 transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Aortic SMC mRNA profiles of 20-week western diet feeding wild type (WT) and miR-21fl/flmTmG/Myh11ERT2 mice