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In JoVE (1)
- RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Other Publications (2)
Articles by Dilyara Cheranova in JoVE
RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells
Dilyara Cheranova, Margaret Gibson, Suman Chaudhary, Li Qin Zhang, Daniel P. Heruth, Dmitry N. Grigoryev, Shui Qing Ye
Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City
This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.
Other articles by Dilyara Cheranova on PubMed
RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin
PloS One. 2012 | Pubmed ID: 22359579
The dysregulation of vascular endothelial cells by thrombin has been implicated in the development of a number of pathologic disorders such as inflammatory conditions, cancer, diabetes, coronary heart disease. However, transcriptional regulation of vascular endothelial cells by thrombin is not completely understood. In the present study, Illumina RNA-seq was used to profile the transcriptome in human pulmonary microvascular endothelial cells (HMVEC-L) treated with thrombin for 6 h to gain insight into thrombin's direct effects on the endothelial function. Out of 100 million total reads from a paired end sequencing assay, 91-94% of the reads were aligned to over 16,000 genes in the reference human genome. Thrombin upregulated 150 known genes and 480 known isoforms, and downregulated 2,190 known genes and 3,574 known isoforms by at least 2 fold. Of note, thrombin upregulated 1,775 previously unknown isoforms and downregulated 12,202 previously unknown isoforms by at least 2 fold. Many genes displayed isoform specific differential expression levels and different usage of transcriptional start sites after the thrombin treatment. The cross comparisons between our RNA-seq data and those of DNA microarray analysis of either 6 h thrombin treated HUVEC or 5 h TNFα treated HMVEC have provided a significant overlapping list of differentially expressed genes, supporting the robust utility of our dataset. Further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying thrombin mediated endothelial dysfunction in various diseases and provide new leads of potential therapeutic targets.
Pleiotropic Functions of Pre-B-cell Colony-enhancing Factor (PBEF) Revealed by Transcriptomics of Human Pulmonary Microvascular Endothelial Cells Treated with PBEFsiRNA
Genes to Cells : Devoted to Molecular & Cellular Mechanisms. May, 2012 | Pubmed ID: 22487217
This study profiled transcriptomes of human pulmonary microvascular endothelial cells (HMVEC-L) treated with pre-B-cell colony-enhancing factor (PBEF) siRNA or scrambled RNA to gain insight into transcriptional regulations of PBEF on the endothelial function using the Affymetrix GeneChips HG-U133 plus 2. Several important themes are emerged from this study. First, PBEF affected expressions of multiple genes in the endothelium. Expression of 373 genes was increased and 64 genes decreased by at least 1.3-fold in the PBEFsiRNA-treated HMVEC-L versus the scramble RNA control. Second, the microarray results confirmed previous reports of PBEF-mediated gene expressions in some pathways but provided a more complete repertoire of molecules in those pathways. Third, most of the affected canonical pathways have not previously been reported to be PBEF responsive. Fourth, network analysis supports that PBEF has pleiotropic functions. Our first transcriptome analysis of human pulmonary microvascular endothelial cells treated with PBEFsiRNA has provided important insights into the transcriptional regulation of gene expression in HMVEC-L cells by PBEF. Further in-depth analysis of these transcriptional regulations may shed light on molecular mechanisms underlying PBEF-mediated endothelial functions and dysfunctions in various diseases and provide new leads of therapeutic targets to those diseases.