P5394Whole transcriptome sequencing to define monocyte subpopulation-specific lncRNA profiles in acute myocardial infarction patients and healthy controls

Abstract Background Monocytes are important immune cells in both onset and resolution of inflammation during pathologies such as acute myocardial infarction (AMI) and atherosclerosis. Long non-coding RNAs (lncRNAs) have emerged as novel regulatory and highly cell-specific molecules that can modulate cell physiology in numerous ways such as mRNA (de-)stabilization, micro RNA sponging or scaffolding of RNA binding proteins. Purpose Define a complete human monocyte subpopulation-specific transcriptome of long non-coding RNAs and characterize the difference in the profile of these RNA molecules in AMI patients. Methods and results Human monocyte subpopulations, defined as classical, intermediate and non-classical based on the expression of the surface markers CD14 and CD16, were collected on a FACS Aria II. Ribosomal-depleted cDNA libraries generated from total RNA were processed for Next Generation Sequencing on a HiSeq Illumina 2000. Computationally intensive bioinformatics revealedannotated lncRNAs, antisense, pseudogene and circular RNAs with significant difference in their expression profiles within subpopulations of healthy donors such as MEG3 or TERC, potential role players in cardiovascular disease. Our data also unraveled novel non-annotated ncRNAs not yet reported to reference databases, which are expected to be monocyte-specific. We applied certain criteria to identify potential candidate molecules such as annotation with existing Ensembl ID and a pre-determined expression level. Thereupon we selected differentially regulated long non-coding RNAs differentially expressed in cardiovascular disease and discovered 18 annotated potential lncRNAs dysregulated in classical monocytes of AMI patients such as HLX antisense, which might be involved in monocyte differentiation. Additional 5 targets appeared specific only for differences in intermediate and 3 with additional specific differences only in non-classical monocytes.Real-time PCR was applied for validation of long non-coding linear and circular RNAs differential expression and also to determine their nucleocytoplasmic distribution. We observed preferential nuclear expression for most lncRNAs in contrast to cytoplasmic circRNAs. In vitro assays for silencing and overexpressing certain target molecules as well astreatment withinflammatory stimuli and in silicoanalysis with different bioinformatics tools such as FANTOM and UCSC browser will help to unravel their functionality. Conclusions Next generatio