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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and miR-21-/- Smooth Muscle Cell Transcriptomes

ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. 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 function at basal level. Methods: Aortic smooth muscle cell (SMC) mRNA profiles of 12-week-old wild-type (WT) and SMC specific miR-21 knockout (miR-21fl/flmTmG/Myh11ERT2) mice 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 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the SMCs of WT and miR-21fl/flmTmG/Myh11ERT2 mice. RNA-seq data revealed >400 genes differentially expressed in miR-21 knockout SMCs. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized pathways that may contribute to SMC function. 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: GSE183830 | 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.

Project description:Smooth muscle cells (SMCs) display dynamic plasticity by changing phenotype under various pathophysiological conditions. Uncovering how SMCs regulate their phenotype is a key to understanding the molecular mechanisms of a number of gastrointestinal diseases. MicroRNAs (miRNAs), generated in cells by Dicer, have been identified as regulators of the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal SMCs in a transgenic animal model. We generated SMC-specific Dicer (smDicer) null animals that express the reporter, green fluorescence protein (eGFP), in a SMC-specific manner. eGFP labeled SMCs were used for morphological, cytometric and genetic studies of smDicer null mutant and wild type control mice. The structure of the bowel wall was examined by confocal microscopy, and function was evaluated by recording spontaneous and evoked contractions. SMCs were purified with fluorescence-activated cell sorting and SM specific gene expression studies were performed with genechip arrays, PCR and quantitative PCR. Bioinformatic analyses were used to characterize the interaction between miRNAs and target genes. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs. Profiling and bioinformatic analyses showed SMC phenotype is regulated by a complex network of positive and negative feedback by SM miRNAs, serum response factor (SRF), and other transcriptional factors. SM miRNAs play an important role in growth, development and survival of SMCs. Phenotypic changes of SMCs may be regulated through multiple pathways in an interaction network of SM miRNAs, SRF, and additional transcriptional factors. We used microarrays to identify genetic changes during the development of gastric smooth muscle cells in the smDicer null mice. Mouse mRNA microarrays were performed on GeneChipÂ® Mouse Genome 430 2.0 Arrays (Affymetrix). Total RNAs isolated from the jejunal muscularis of the smDicer knockout (KO) and the wild type (WT) control mice at the ages of ~3 weeks were used for the arrays. The cDNA synthesis, hybridization, cDNA labeling, and scanning were performed. Gene expression between the smDicer KO and the WT controls were compared.

Project description:Tissue Factor Pathway Inhibitor-2 is Induced by Fluid Shear Stress in Vascular Smooth Muscle Cells and Affects Cell Proliferation and Survival Introduction: Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function. Methods: Laminar FSS of arterial level (14 dynes/cm2) was applied to SMC cultures for 24 h in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology and the results further verified by real time (RT) PCR and immunoblotting. Protein expression was also studied in the rat carotid artery after balloon-injury and DNA synthesis and apoptosis was examined in SMCs in vitro. Results: Microarrays identified tissue-pathway inhibitor-2 (TFPI-2) as the most differentially expressed gene by FSS in cultured SMCs. The regulatory effect of FSS on the expression of TFPI-2 was confirmed by RT-PCR and immunobloting demonstrating a more than 400-fold increase in TFPI-2 expression in SMCs exposed to FSS compared to static controls and a consistent upregulation at the protein level. Functionally, SMC proliferation was decreased by FSS and recombinant TFPI-2 was found to inhibit SMC proliferation and induce SMC apoptosis as indicated by activation of caspase-3. In vivo, TFPI-2 expression was found to be up-regulated 5, 10 and 20 h after rat carotid balloon-injury and immunohistochemistry demonstrated TFPI-2 protein in luminal SMCs exposed to FSS in rat carotid intimal hyperplasia 10 days after balloon-injury. Conclusion: FSS strongly influence gene expression in cultured SMCs and induce TFPI-2 expression, which is also expressed after rat carotid balloon injury in luminal SMCs exposed to FSS. Functionally, TFPI-2 may play an important role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 control SMC function. 3 x 2 samples from a paired fluid shear stress experiment on smooth muscle cells.

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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and miR-21-/- Smooth Muscle Cell Transcriptomes

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