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Integrating microRNA-lncRNA-gene regulatory circuits in sprouting angiogenesis Noghero A. (1), Rosano S. (1), Corà D. (1*), Bussolino F. (1*) (1) Dept. of Oncology, University of Torino - Str. Prov. 142 Km. 3.95, I-10060 Candiolo, Torino, Italy * email to: [email protected], [email protected] In vitro model of Sprouting Angiogenesis: HUVEC SPHEROIDS Transcriptome Screening: protein coding genes + lncRNAs + microRNAs SPHV SPHV Protein_coding Ang-1 SPHV FGF-2 SPHC VEGF-A SPHC Ctr SPHC In such matrix, spheroids are induced to form endothelial sprouts by the effect of exogenously added VEGF-A. This model can recapitulate the endothelial cell response triggered by an angiogenic stimulus in a 3D environment. H2D RNA from sprouted spheroids and from control spheroids that were not exposed to VEGF-A stimulus were subjected to RNA sequencing, for both small and long poly-A mRNAs. RNA from HUVEC cultured in 2D basal conditions was also included in this analysis as a reference. High-throughput RNA-sequencing was performed on an Illumina HiSeq 2000 sequencer for small and long-mRNA messengers, with a similar experimental design. H2D We used a model of sprouting angiogenesis consisting in spheroids made of human umbilical vein endothelial cells (HUVEC) cultured in 3D matrix formed by collagen gel. MALAT1 SPHV SPHV SPHV SPHC SPHC SPHC H2D H2D RNA-seq summary of results: mRNAs / lncRNAs / microRNAs circuits SPHV SPHV SPHV SPHC Plasma membrane SPHC Cell migration SPHC Blood vessel development H2D Notch signaling pathway H2D LncRNAs MicroRNAs Protein coding genes lncRNA - Human genome annotation from ENSEMBL 75. - Human microRNA annotation from MIRBASE 18. - RSEM isoform-specific pipeline (100 bps long paired- end reads). - 10168 ENSTs (3681 ENSGs) found differentially expressed with FDR < 0.01. - 183 mature microRNAs found differentially expressed with FDR < 0.01. Cell adhesion EGFR signaling Phosphate metabolsim Differential comparative analysis allowed the investigation for combination of lncRNA / protein coding genes / microRNAs selectively activated or inhibited during the process. Response to wounding Protein_coding mRNA As final result, a network composed by lncRNA / protein coding genes / microRNAs triplets was generated. Edges were superimposed with expression correlation values, thus allowing in particular the identification of global patterns and specific “hub” microRNAs, which we propose for experimental validation. Small molecule biosynthesis Vescicle Extracellular matrix Cell adhesion Cell migration Cell-cycle and Replication lncRNA References: – Judah Folkman, Nature Reviews Drug Discovery 6, 273-286 (2007). – Friard et al, BMC Bionformatics 11:435 (2010). – Pandolfi et al, Cell 147(2):344-57 (2011). – Cora' et al, Trends Mol Med. 20(10):589-98 (2014). – Wang P et al, Nucleic Acids Res. 43(7):3478-89 (2015). Expression profiles for proteing_coding genes, lncRNAs and microRNAs where integrated in several ways. Here, we show the results in the case of pairwise Spearman correlation between all protein_coding vs lncRNAs followed by a Gene Ontology functional analysis. MicroRNA The integration of RNA-seq results with databases of microRNA-mediated regulatory interactions identified several microRNA-lincRNA-gene circuits (triplets) significantly modulated during VEGF-A driven sprounting angiogenesis. Functional analysis of lncRNAs and protein coding genes. We used Gene Set Enrichment Analysis (GSEA) to identify the major biological pathways being activated during the sprouting process. We found a statistically significant positive association with several gene sets representative for the VEGF pathway (internal control), protein translation, extracellular matrix organization and cell adhesion molecules. Furthermore, such analysis identified a modulation of sets of genes related to specific metabolic pathways.