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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE183829 | GEO | 2022/06/10

REPOSITORIES: GEO

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Project description:Purpose: using single cell transcriptome profiling to find cell-type specific genes regulated by microRNA-21 following western diet feeding in atherosclerotic mouse models. Methods: WT/mTmG/ Myh11ERT2 and miR-21fl/flmTmG/Myh11ERT2 littermates were injected Tamoxifen at 5weeks old for CRE recombination. Later received AAV8-PCSK9 injection then fed on western diet (1.25% cholesterol; D12108; Research Diets, Inc) for 20 weeks to induce atherosclerosis. Aortas were harvested from WT/mTmG/ Myh11ERT2 and miR-21fl/flmTmG/Myh11ERT2 mice and digested in 1.5mg/ml collagenase A (10103578001, Roche) + 0.5mg/ml elastase (6365, Worthington) buffer supplemented with 0.1% actinomycin at 37oC for 1h with gentle shaking. Digested aortas were passed through a 70 μm Cell Strainer and stained with a live/dead viability dye eFluor 450 (Thermo Fisher Scientific). Viable eGFP+ (SMCs) and eGFP-/tdTomato+ (non-SMCs aortic cells) were sorted by FACS Aria III (BD Biosciences). The sorted eGFP+ (SMCs) and eGFP-/tdTomato+ (non-SMCs aortic cells) cells were mixed to represent complete profile of aortic cells and then encapsulated into droplets and processed following manufacturer’s specifications using 10X Genomics GemCode Technology. Equal numbers of cells per sample were loaded on a 10x Genomics Chromium controller instrument to generate single-cell Gel Beads in emulsion (GEMs) at Yale Center for Genome Analysis. Lysis and barcoded reverse transcription of polyadenylated mRNA from single cells were performed inside each GEM followed by cDNA generation using the Single Cell 3’ Reagent Kits v2 (10X Genomics). Libraries were sequenced on an Illumina HiSeq 4000 as 2 × 100 paired-end reads. Results: 5404 cells from WT/mTmG/ Myh11ERT2 sample and 6911 cells from miR-21fl/flmTmG/Myh11ERT2 sample for expression analysis and distinguished phenotypes were identified for SMCs lacking miR-21. Following pathway analysis on the DEGs revealed pathways regulated by miR-21 deficiency in SMCs that contributes to advancing atherosclerotic plaque. Conclusions: Our study represents the first detailed transcriptomic analysis of atherosclerotic aortas from SMC specific knockout of miR-21. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that when lacking miR-21, a subset of SMCs are prone to apoptosis that supported the overall pro-atherogenic phenotype by miR-21 deficiency.

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

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