RNA sequencing of porcine smooth muscle cells, after treatments with platelet derived growth factor and S100A4 protein
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ABSTRACT: Smooth muscle cells were treated with platelet derived growth factor (PDGF-BB) and S100A4 protein to decipher the mechanisms by which they contribute to smooth muscle cell phenotypic transition. We report that PDGF-BB treatment upregulates genes related to growth response or to extracellular matrix component proteases, while S100A4 upregulates pro-inflammatory genes. When used in combination, PDGF-BB and S100A4 show synergistic action by enhancing the upregulation of genes already affected by S100A4 and by inducing the upregulation of genes exclusive for this condition.
Project description:To explore global molecular changes in smooth muscle in response to PDGFR activation, primary human bladder smooth muscle cells were treated with 1 nM PDGF-BB (hereafter PDGF) for 0, 4 or 24 h. Total RNA were prepared, and analyzed using expression profiling, and subjected to bioinformatic and functional interrogation. To identify molecular signatures of bladder smooth muscle peturbed by PDGF, primary human bladder smooth muscle cells were treated with 1 nM PDGF-BB (hereafter PDGF) for 0, 4 or 24 h.
Project description:To further understand the mechanism approach to occlusive vein grafts, we have employed RNA-seq to identify key genes in occlusive venin grafts following CABG in human. The occluded vein graft and intraoperative spare great saphenous vein of patients undergoing clinical re-coronary artery bypass grafting were obtained for RNA-seq. The sequencing results were cleaned and bioinformatics annlysis was conducted by using WGCNA and a variety of online databases and software. The key genes or proteins affecting the occurrence of vein graft restenosis were screened out(ITGB2), and the expression level of the target genes or proteins was verified by real-time PCR and Western blot. And to further learn the effect of ITGB2 in human primary venous smooth muscle cells, ITGB2 gene was silenced by SiRNA. The effect of ITGB2 silencing on proliferation, migration and invasion of venous smooth muscle cells after PDGF-BB-stimulation were detected by Edu assay, scrathch experiment and transwell experiment. And Edu assay showed that ITGB2 silencing could inhibit the proliferation of PDGF-BB sitmulated smooth muscle cells. Scratch assay showed that ITGB2 silencing inhibited the migration of PDGF-BB stimulated smooth muscle cells. Transwell assay showed that ITGB2 silencing significantly inhibited the invasion of PDGF-BB stimulated smooth muscle cells. It is indicated that ITGB2 was the key gene in vein graft restenosis,and may be the potential treatment target in restenosis patients.
Project description:We report the application of small RNA sequencing for high-throughput profiling of small RNA under 75 bp in vascular smooth muscle cell. By a reading depth of 30M and single stranded sequencing, we generated the small RNA signature on differentiated and de-differentiated vascular smooth muscle cell induced by PDGF-BB and H3K4me2 editing. We found that PDGF-BB and H3K4me2 editing induced de-differentiation modulated miRNA profile significantly, which was demonstrated at least in part responsible for modulated vascular smooth muscle cell phenotype.
Project description:In order to further study the role of circular RNA in the phenotypic transformation of vascular smooth muscle cells (VSMCs), the differential expression profile of circRNA in the phenotypic transition of VSMCs induced by platelet-derived growth factor-BB (PDGF-BB) was screened using chip technology. Vascular smooth muscle cells from rat thoracic aorta were induced with 20ng/ml PDGF-BB as the experimental group and compared with the control group. After induction for 24 hours, the differentially expressed circRNA was screened by circular RNA chip.
Project description:To explore global molecular changes in smooth muscle in response to PDGFR activation, primary human bladder smooth muscle cells were treated with 1 nM PDGF-BB (hereafter PDGF) for 0, 4 or 24 h. Total RNA were prepared, and analyzed using expression profiling, and subjected to bioinformatic and functional interrogation.
Project description:Comparison of gene expression between Stat1+/- and Stat1-/- dermal fibroblasts or vascular smooth muscle cells after treatment with PDGF-BB for one hour.
Project description:SILAC analysis of human primary bladder smooth muscle cells treated with platelet-derived growth factor for 0, 4, and 24 h. Platelet-derived growth factor-BB (PDGF-BB) is a mitogen and motogen that has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells (SMC) that characterize pathologic tissue remodeling in hollow organs. To explore the signals induced by PDGF on a global scale, we performed expression profiling and quantitative proteomics analysis of PDGF-treated human visceral SMC. 1695 genes and 241 proteins were identified as differentially expressed in PDGF-treated primary bladder SMC versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB and RUNX1 as the transcription factors most significantly networked with upregulated genes; DDIT3, NFAT5, and SOX5 were most networked with downregulated genes. For protein identification and quantification, raw mass spectrometric data were analyzed with MaxQuant software (version 1.0.13.13). The parameters were set as follows. In the Quant module, SILAC triplets was selected; oxidation (M) and acetyl (Protein N-term) were set as variable modification; carbamidomethyl (C) was set as fixed modification; concatenated IPI human database (version 3.52) (74,190 forward sequences and 74,190 reverse sequences) was used for database searching; all other parameters were default. Tandem mass spectra were searched by Mascot (version 2.2.0.4) (Matrix Science, Boston, MA). In the Identify module, all parameters were default, except that maximal peptide posterior error probability was set as 0.05. False discovery rates for protein and peptide identifications were both set at 0.01.
Project description:We analyzed differentially expressed genes in mouse aortic smooth muscle cells (MASMCs) from wild-type or Hint1-/- mice treated with 20ng/ml PDGF-BB for 24h to explore the mechanism that HINT1 triggers VSMC phenotypic switching.
Project description:Crotonylation of histones is discovered of late as one of post-translational modification that can regulate gene expression. However, the function of crotonylation on non-histone proteins in vascular smooth muscle cells (VSMC) is unclear. Here, we aim to use modification and proteomic analysis to find the cellular characteristic of crotonylated non-histone proteins and the crosstalk with ubiquitinated proteins in vascular smooth muscle cell (VSMC) phenotypic remodeling. We performed modification and proteomic analysis of VSMCs before and after stimulated with platelet-derived growth factor-BB (PDGF-BB). The crotonylated and ubiquitinated pan-antibody was used to enrich the protein and then subjected to high-throughput mass spectrometry analysis. The enrichment analysis was performed within differentially modified proteins in regards to GO terms, KEGG and protein domain.
Project description:Pulmonary hypertension (PH) patients typically present with a diminished platelet count, but the role of platelets in the development and progression of PH remains unclear.Transcriptomic analysis revealed that platelets from PH patients exhibited an upregulation of genes associated with cellular adhesion, platelet activation, and adhesion. Notably, the hub genes, glycoprotein IIb/IIIa (GP IIb/IIIa), were implicated in mediating platelet-endothelium adhesion through their interaction with intercellular adhesion molecule-1 (ICAM-1) on pulmonary artery endothelial cells, triggering platelet activation and the subsequent release of platelet-derived growth factor BB (PDGF-BB).