Transcript Poster_EMBO_Weizmann_2016_v1 - INFN

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.