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Deep-Sequencing of Endothelial Cells Exposed to Hypoxia Reveals the Complexity of Known and Novel Micrornas

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Deep-Sequencing of Endothelial Cells Exposed to Hypoxia Reveals the Complexity of Known and Novel Micrornas Downloaded from rnajournal.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs CHRISTINE VOELLENKLE,1,6 JEROEN VAN ROOIJ,1,6 ALESSANDRO GUFFANTI,2,6 ELENA BRINI,2 PASQUALE FASANARO,3 ELEONORA ISAIA,1 LARRY CROFT,4 MATEI DAVID,5 MAURIZIO C. CAPOGROSSI,3 ANNA MOLES,2 ARMANDO FELSANI,2 and FABIO MARTELLI3,7 1Laboratorio di Cardiologia Molecolare, IRCCS-Policlinico San Donato, San Donato Milanese, 20097 Milan, Italy 2Genomnia srl, Lainate, 20020 Milan, Italy 3Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata-IRCCS, 00167 Rome, Italy 4Institute for Molecular Bioscience, University of Queensland, 4072 Brisbane, Australia 5Computational Biology Laboratory, University of Toronto, M5S 3G4 Toronto, Canada ABSTRACT In order to understand the role of microRNAs (miRNAs) in vascular physiopathology, we took advantage of deep-sequencing techniques to accurately and comprehensively profile the entire miRNA population expressed by endothelial cells exposed to hypoxia. SOLiD sequencing of small RNAs derived from human umbilical vein endothelial cells (HUVECs) exposed to 1%% O2 or normoxia for 24 h yielded more than 22 million reads per library. A customized bioinformatic pipeline identified more than 400 annotated microRNA/microRNA* species with a broad abundance range: miR-21 and miR-126 totaled almost 40%% of all miRNAs. A complex repertoire of isomiRs was found, displaying also 59 variations, potentially affecting target recognition. High- stringency bioinformatic analysis identified microRNA candidates, whose predicted pre-miRNAs folded into a stable hairpin. Validation of a subset by qPCR identified 18 high-confidence novel miRNAs as detectable in independent HUVEC cultures and associated to the RISC complex. The expression of two novel miRNAs was significantly down-modulated by hypoxia, while miR- 210 was significantly induced. Gene ontology analysis of their predicted targets revealed a significant association to hypoxia- inducible factor signaling, cardiovascular diseases, and cancer. Overexpression of the novel miRNAs in hypoxic endothelial cells affected cell growth and confirmed the biological relevance of their down-modulation. In conclusion, deep-sequencing accurately profiled known, variant, and novel microRNAs expressed by endothelial cells in normoxia and hypoxia. Keywords: microRNA; isomiR; deep-sequencing; hypoxia; endothelium INTRODUCTION a ribonucleoprotein complex mediating post-transcriptional gene silencing. Sequence-specific interaction with the func- MicroRNAs (miRNAs) are endogenous, short, 21- to tional miRNA leads to destabilization or translational in- 23-nucleotide (nt), nonprotein-coding RNAs that act as neg- hibition of the target mRNAs by the Ago protein (Winter et al. ative regulators of gene expression, modulating the trans- 2009). For several pre-miRNAs, both hairpin arms are lational efficiency and/or the stability of target messenger processed and give rise to a functional miRNA. In this case, RNAs (mRNAs). These RNA molecules are transcribed as mature miRNAs are designated with the suffix ‘‘-3p’’ or ‘‘-5p,’’ a long primary transcript, termed pri-miRNA, sequentially depending on the originating hairpin arm. Alternatively, if processed by Drosha and Dicer ribonucleases, first to a pre- either one is expressed at much lower levels, it is designated as cursor-miRNA with a characteristic hairpin structure and miRNA* (star form) (Winter et al. 2009; Krol et al. 2010). then to the mature miRNA species. Then, the single-stranded, MiRNAs have essential functions in physiological pro- mature miRNA is loaded together with Argonaute (Ago) cesses, including cellular differentiation, development, and proteins into the RNA-induced silencing complex (RISC), apoptosis, but are also involved in pathological processes, like cancer (Garzon et al. 2009; McCormick et al. 2010) or cardiovascular diseases (Urbich et al. 2008; Dorn 2010; 6These authors contributed equally to this work. Fasanaro et al. 2010). 7 Corresponding author. Hypoxia describes the insufficient oxygen supply to tissues E-mail [email protected]. Article published online ahead of print. Article and publication date are and occurs during several physiopathological circumstances at http://www.rnajournal.org/cgi/doi/10.1261/rna.027615.111. such as rapid tissue growth, acute and chronic ischemia, RNA (2012), 18:00–00. Published by Cold Spring Harbor Laboratory Press. 1 Downloaded from rnajournal.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Voellenkle et al. organ and tumor development, and high altitude (Semenza owing to the low probability of being miRNA derived. Then, 2010b). Decreased oxygen tension triggers a distinct pro- all remaining reads were trimmed to 25 nt, the maximum gram of responses aimed at relieving tissue hypoxia and expected length for a miRNA (Winter et al. 2009; Krol et al. removing irreversibly damaged cells. These responses include 2010), and used for mapping against various coding and endothelial cell proliferation, migration, and angiogenesis noncoding transcript and genomic data sets. and also growth arrest and apoptotic cell death (Semenza 2010b). Several reports have demonstrated that specific Identification and quantification of annotated miRNAs are involved in the hypoxic response (Ivan et al. miRNAs 2008). In particular, we and others found that the expression of miR-210 is induced in hypoxic endothelial The customized analysis pipeline represented in Figure 1 cells (Fasanaro et al. 2008; Chan et al. 2009), stimulating was used to quantify previously known (annotated) miR- their vascular endothelial growth factor (VEGF)–driven NAs. The reads corresponding to annotated miRNAs were migration and tubulogenesis (Fasanaro et al. 2008). identified by selecting all the reads mapping against the In order to understand how miRNAs regulate gene human sequences contained in miRBase v14.0, excluding expression in endothelial cells exposed to low-oxygen any read mapping also against a collection of other small tension, it is essential to accurately profile the entire miRNA RNAs databases (transfer RNAs [tRNAs], small nucleolar population. Currently, there are three widely used platforms RNAs [snoRNAs], ribosomal RNAs [rRNAs]). Then, fur- for miRNA profiling: quantitative polymerase chain reac- ther selection criteria based on alignment length, absence of tion (qPCR), microarray hybridization (Shendure 2008), and mismatches in the seed region, and absence of sequencing deep-sequencing (also named next-generation sequencing errors were applied: An average of 1.2 million reads per or massively parallel sequencing). While qPCR and micro- library were identified as annotated miRNAs, representing arrays rely on the availability of miRNA sequences in public 305 mature and 96 miRNA* species. As expected, a very and proprietary databases for designing probes or primers significant correlation between the results obtained analyzing (Griffiths-Jones et al. 2008), deep-sequencing offers the pos- the two normoxic and the two hypoxic libraries was observed sibility to comprehensively assess miRNA expression in a (Supplemental Fig. S1). The counts per miRNA were then timely and cost-effective fashion. Along with unprecedented normalized with respect to the total library size, and all scale and linear range, the sequencing approach does not have thermodynamic biases usually associated with hybridization- based qPCR and microarray platforms (Shendure 2008). More- over, deep-sequencing allows discovery of previously unchar- acterized miRNA species. These include not only new miRNAs that eluded previous cloning but also a panoply of miRNA variants, also known as isomiRs (Landgraf et al. 2007; Morin et al. 2008; Fernandez-Valverde et al. 2010). In this study the power of deep-sequencing technology was adopted to determine the complete repertoire of miRNAs expressed by human endothelial cells and to identify their modulations induced by low-oxygen tension. RESULTS Small RNA library generation, sequencing, and primary analysis Human umbilical vein endothelial cells (HUVECs) were cultured either in normoxic conditions or at 1% oxygen for 24 h. Then, small RNA libraries were prepared and deep- sequenced by using a SOLiD3 Plus Sequencer platform. As customary for these kinds of studies, two independent FIGURE 1. Analysis pipeline of annotated miRNAs. Flowchart de- scribing data analysis pipeline designed for the identification of populations were analyzed, each both in normoxia and in mature and star sequences of annotated miRNAs, their isoforms, hypoxia. The corresponding four cDNA libraries yielded a and novel miRNA star sequences. The number of remaining reads/ total of 114 million sequenced reads, and 78% (89 million, hits/miRNAs after each filtering step, averaged across all four libraries, an average of 22.2 million per library) of these reads passed is indicated in italics. The high-stringency filter of alignment (step 5) consisted in minimum alignment length of 17 nt, with a maximum of the quality control filter (quality value [QV] $ 9). Reads one mismatch and three sequencing errors. Reads with sequencing mapping across genomic sequence by >25 nt were removed errors in the first 15 nt or mismatches in the seed region were excluded. 2 RNA, Vol. 18, No. 3 Downloaded from rnajournal.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press microRNA
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