Abstract 14249: Profiling and Discovery of microRNAs Expressed in Hypoxic Endothelial Cells by Massively Parallel Sequencing
Background: MicroRNA profiling, annotation and discovery through massively parallel (deep) sequencing is a very active field in modern functional genomics. However, few examples exist of its application in the cardiovascular arena. Here we describe the high throughput comprehensive analysis of the microRNAs expressed by endothelial cells exposed to hypoxia.
Methods and Results: Total RNA was extracted from Human Umbelical Vein Endothelial Cells (HUVEC) exposed to 1% O2 or normoxia for 24 hrs. Then, RNA was size fractionated and 4 small RNA libraries were prepared and amplified by emulsion PCR. Sequencing-by-ligation was performed using the ABI SOLiD platform, followed by bioinformatic analysis by a customized pipeline using the SHRiMP software. More than 20 millions of mappable reads were obtained for each library. Alignment versus miRBase identified >600 different microRNAs expressed in HUVEC, with reads for each RNA species ranging from 1 to hundreds of thousands. Indeed, the 2 top-abundance transcripts (miR-21 and -126) comprised 40% of total HUVEC microRNAs. Comparison of HUVEC miRNA profiles by qPCR and microarray revealed the superior dynamic range and enhanced specificity of RNA sequencing. Hypoxia/normoxia comparison, followed by qPCR validation (n=5), allowed the identification of 1 down-modulated (miR-7) and 3 up-regulated (miR-150, -210 and -720) previously known microRNAs. Bioinformatic analysis identified 20 not previously described microRNA-star sequences and 994 novel microRNA candidates. After a careful ranking based on read abundance, number and position of genomic mappings, position of the sequence in the stem of the predicted hairpin and confirmation with a second genomic mapping software (ABI Bioscope rmapper), we identified 16 high-confidence new microRNAs.
Conclusions: Massively parallel smallRNA sequencing accurately profiled known and novel microRNAs expressed by endothelial cells in the presence or absence of hypoxia.
- © 2010 by American Heart Association, Inc.