Identification and Initial Functional Characterization of a Human Vascular Cell–Enriched Long Noncoding RNA

Abstract

Objective Long non-coding RNAs (lncRNAs) represent a rapidly growing class of RNA genes with functions related primarily to transcriptional and post-transcriptional control of gene expression. There is a paucity of information about lncRNA expression and function in human vascular cells. Thus, we set out to identify novel lncRNA genes in human vascular smooth muscle cells and to gain insight into their role in the control of smooth muscle cell phenotypes. Approach and Results RNA-sequencing of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell-enriched lncRNA we call SENCR (Smooth muscle and Endothelial cell enriched migration/differentiation-associated long Non-Coding RNA). Strand-specific RT-PCR and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5’ end of the FLI1 gene and exists as two splice variants. RNA fluorescence in situ hybridization and biochemical fractionation studies demonstrate SENCR is a cytoplasmic lncRNA. Consistent with this observation, knockdown studies reveal little to no cis-acting effect of SENCR on FLI1 or neighboring gene expression. RNA-sequencing experiments in smooth muscle cells following SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, while a number of pro-migratory genes are increased. RT-PCR and Western blotting experiments validate several differentially expressed genes following SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration. Conclusion SENCR is a new vascular cell-enriched, cytoplasmic lncRNA that appears to stabilize the smooth muscle cell contractile phenotype.

Long Noncoding RNAs in Atherosclerosis

“…Using of RNA-sequencing studies, Bell et al 65) revealed 31 unannotated lncRNAs from human coronary artery smooth muscle cells and investigated a lncRNA SENCR (smooth muscle and endothelial cell enriched migration/differentiation-associated lncRNA). SENCR knockdown decreased myocardin al 72) identified a dendritic cell-specific lncRNA termed lnc-DC playing a critical role in the differentiation of dendritic cells from human monocytes and mouse bone marrow, a cell type playing important roles in atherosclerosis 73) .…”

“Going Long”: Long Non-Coding RNAs as Biomarkers

“…5 lncRNAs display high organ and cell specificity and are involved in maintaining cell integrity. Previous analyses of cardiovascular lncRNAs were performed in cultured cells, [6][7][8] animal models, 9,10 and human heart biopsies. 10,11 Circulating lncRNAs have been suggested as potential biomarkers for cancer, but few studies have explored lncRNAs as biomarkers in the context of cardiovascular disease.…”

“…[6][7][8]12,15 With the exception of MIAT, all analyzed lncRNAs were differentially expressed in patients with MI: antisense hypoxia inducible factor 1α, KCNQ1OT and MALAT1 were upregulated, whereas ANRIL was downregulated. MALAT1 and antisense hypoxia inducible factor 1α are known to be induced under hypoxia.…”

Function and Therapeutic Potential of Noncoding RNAs in Cardiac Fibrosis

“…72 Contrarily, the finding that their promoters are often highly conserved 73 and their expression is AAV9 indicates adeno-associated viruses; AngII, angiotensin II; CTGF, connective tissue growth factor; Fbln2, fibullin-2; FoxO3, forkheadbox O3; MeCP2, methyl-CpG-binding protein 2; MHC, myosin heavy chain; MI, myocardial infarction; PTEN, phosphatase and tensin homolog; RasGAP, Ras-GTPase activating protein; SPRY, Sprouty homologue 1; TAC, transverse aortic constriction; and TGFβ, transforming growth factor-β. by guest on May 10, 2018 http://circres.ahajournals.org/ Downloaded from generally more tissue-specific than protein-coding genes does suggest a functional relevance for at least some of them. 74 In fact, functional studies have revealed that lncRNAs participate in a variety of biological processes, including transcriptional regulation by recruiting chromatin regulatory proteins to specific genomic locations, genomic imprinting, organization of protein complexes, and shaping distinct nuclear structures.…”

“…83 In endothelial cells and smooth muscle cells, several lncRNAs (ie, MALAT1 and SENCR) have been shown to control migration and differentiation. 84,85 In fibroblasts, TERRA lncRNAs (eg, telomeric repeat-containing RNA) are implicated in modulating the structure and processing of deprotected telomeres. 86,87 Deprotected telomeres elicit a DNA damage response leading to chromosome instability, which is in turn associated with cellular senescence.…”