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: davide.cora@ircc.it, federico.bussolino@ircc.it

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).

In vitro model of Sprouting Angiogenesis:HUVEC SPHEROIDS

Ctr VEGF-A

FGF-2 Ang-1

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.

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.

Transcriptome Screening:protein coding genes + lncRNAs + microRNAs

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 H2D SPHC SPHC SPHC SPHV SPHV SPHV

Protein_coding

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.

RNA-seq summary of results:

- 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.

lncRNA MicroRNA

H2D H2D SPHC

SPHC SPHC SPHV SPHV SPHV

H2D H2D SPHC

SPHC SPHC SPHV SPHV SPHV

MALAT1

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 functionalanalysis.

lncRNA

Protein_coding mRNA

Cell-cycle andReplication

Cell adhesionCell migration

EGFR signaling

Phosphate metabolsim

Small moleculebiosynthesis

Vescicle

Response to wounding

Extracellular matrix

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.

Differential comparative analysis allowed the investigation for combination of lncRNA / protein coding genes / microRNAs selectively activated or inhibited during the process.

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.

Functional analysis of lncRNAs and protein coding genes.

mRNAs / lncRNAs / microRNAs circuits

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 metabolicpathways.

LncRNAsMicroRNAsProtein coding genes

Notch signalingpathway

Blood vesseldevelopment

Cellmigration

Plasmamembrane

Celladhesion