Single cell RNA Sequencing Facilitates understanding of cell-type specific function of miR-21 during Atherosclerosis

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE188978 | GEO | 2022/06/10

REPOSITORIES: GEO