Peritoneal fluid modifies the miRNA expression profile in

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Peritoneal fluid modifies the miRNA expression profile in
endometrial and endometriotic cells from women with
endometriosis.
Josep Marí-Alexandre1, Salam Salloum-Asfar2, Ana Belén Arroyo2, Rocío González-Conejero2,
Vicente Vicente2, Moisés Barceló1, Javier García-Oms3, Amparo Estellés1, Juan GilabertEstellés3, Constantino Martínez2, Aitana Braza-Boils1.
1 Grupo
de Hemostasia, Trombosis, Aterosclerosis y Biología Vascular. Instituto de
Investigación Sanitaria La Fe, Valencia (Spain). 2Centro Regional de Hemodonación. Servicio
de Hematología y Oncología Médica. Hospital Universitario Morales Meseguer. Universidad de
Murcia (Spain). IMIB. 3Área Maternoinfantil. Hospital General Universitario, Valencia (Spain).
Backgroun
d
ENDOMETRIOSIS, characterized by endometrial-like tissue outside the uterus,
has been associated with an aberrant angiogenesis (McLaren, 2000)
Retrogade menstruation theory
(J. A. Sampson , 1920s)
Although 90% of women present retrograde menstruation,
only 10% develop endometriosis
“ The important thing is not the apparition of endometrial fragments in the peritoneum,
(…) but the alterations that allow some implants to disappear and others to persist and
proliferate”
(Koninkx, 1999)
Backgroun
d
Peritoneal fluid
Interest for their study:
Ectopic lesions located in the pelvic peritoneum are influenced by this fluid.
CURRENT RESEARCH IN THE FIELD
Increased levels of
protein oxidative stress
markers
(Santulli et al. 2014)
Elevated levels of
pro-inflammatory
cytokines
(Rakhila et al. 2014)
Presence of NET
formation
(Berkes et al. 2014)
COMPOSITION
Macrophag
es
Peritoneal Fluid
ENDOMETRI
AL CELLS
Red blood
cells
Small molecules
diffused from
plasma
Ovarian
hormone
s
Growth
factors
Backgroun
d
Several authors have suggested the contribution of a dysregulated microRNA
(miRNA) expression in the development of this condition.
Backgroun
d
miRNAs are small non-coding RNAs (19-22 nt) that bind to target mRNAs and
mediate translational repression or mRNA degradation.
Complexity in understanding the regulatory mechanisms
Backgroun
d
Backgroun
d
“One for all and all
for one”
Backgroun
d
If the expression of a given miRNA is pathologically up-regulated, ALL its target
mRNAs will be repressed,
… and the opposite if this miRNA is down-regulated.
Objective
Our aim was to assess the effect of peritoneal fluid from patients on the
miRNA expression profile in endometrial and endometriotic cells from
patients.
To investigate the targets of the most dysregulayted miRNAs in
response to peritoneal fluid from patients
Methods
Peritoneal fluid
(Proliferative
poolsphase)
Control women
(n=10)
Endometriosis
patients
(n=10)
CPF Pool
EPF Pool
Tissues for primary cell
Control women
Endometriosis patients
cultures
Control endometria
(n=8)
Eutopic
Ovarian
endometria endometriom
(n=11)
a
(n=11)
Endometrial stromal
cell culture
!  CPF: control peritoneal fluid.
!  EPF: endometriotic peritoneal fluid.
Cell
growth
Pre-treatment
Treatment
DMEM without FBS/ 4h
Processi
ng
With EPF/CPF Pool or without PF (LP0)/4h
Cosín et al, Hum. Reprod., 2010
Braza-Boïls, Plos One, 2013
Methods
Peritoneal fluid
(Proliferative
poolsphase)
Control women
(n=10)
Endometriosis
patients
(n=10)
CPF Pool
EPF Pool
Control women
cultures
Control endometria
(n=8)
Eutopic
Ovarian
(n=11)
a
(n=11)
Endometrial stromal
cell culture
Cell
growth
Pre-treatment
Treatment
Processi
ng
Methods
Peritoneal fluid
(Proliferative
poolsphase)
Control women
(n=10)
Endometriosis
patients
(n=10)
CPF Pool
EPF Pool
Control women
cultures
Control endometria
(n=8)
Eutopic
Ovarian
(n=11)
a
(n=11)
Endometrial stromal
cell culture
Cell
growth
Pre-treatment
Treatment
Processi
ng
Methods
Cells
VEGF-A
quantification
Supernatants
Total RNA extraction
miRNA Arrays
Control
end.
In silico studies
of differently expressed
miRNAs
(p<0.05; ±2 fold-change
ANOVA)
Angiogene
miRNA selection:
miR-16-5p
miR-21-5p miR-130a-5p
miR-29c-3p miR-149-5p
miR-185-5p
miR-106b-5p miR-195-5p
miR-424-5p
LPE
LPC n=9
LP0
LPE
Eutopic end. LPCn=12
LP0
LPE
Ovarian LPC n=9
endma.
LP0
qRT-PCR validation
sis
Proteolysis
Angiogene
sis
Functional
experimen
ts
Luciferase
assays
Methods
Cells
VEGF-A
quantification
Supernatants
miRNA Arrays
Control
end.
In silico studies
miRNAs
ANOVA)
Angiogene
miRNA selection:
miR-16-5p
miR-185-5p
miR-424-5p
LPE
LPC n=9
LP0
LPE
LP0
LPE
Ovarian LPC n=9
endma.
LP0
qRT-PCR validation
sis
Proteolysis
Angiogene
sis
Functional
experimen
ts
Luciferase
assays
Methods
Cells
VEGF-A
quantification
Supernatants
miRNA Arrays
Control
end.
In silico studies
miRNAs
ANOVA)
Angiogene
miRNA selection:
miR-16-5p
miR-185-5p
miR-424-5p
LPE
LPC n=9
LP0
LPE
LP0
LPE
Ovarian LPC n=9
endma.
LP0
qRT-PCR validation
sis
Proteolysis
Angiogene
sis
Functional
experimen
ts
Luciferase
assays
Methods
Cells
VEGF-A
quantification
Supernatants
miRNA Arrays
Control
end.
In silico studies
miRNAs
ANOVA)
Angiogene
miRNA selection:
miR-16-5p
miR-185-5p
miR-424-5p
LPE
LPC n=9
LP0
LPE
LP0
LPE
Ovarian LPC n=9
endma.
LP0
qRT-PCR validation
sis
Proteolysis
Angiogene
sis
Functional
experimen
ts
Luciferase
assays
Pricipal
Component
Analysis
Results
Arrays of stimulated cell
cultures
Control cells
Eutopic cells
0PF
CPF
Ectopic cells
EPF
GeneChip miRNA Arrays (Affymetrix)
Pricipal
Component
Analysis
Results
cultures
Control cells
Eutopic cells
0PF
CPF
Ectopic cells
EPF
Pricipal
Component
Analysis
Results
cultures
Control cells
Eutopic cells
0PF
CPF
Ectopic cells
EPF
Results
V u l c a n o
Plots
cultures
Eutopic cells
Log2(fold-change)
P-value
Log2(fold-change)
Log2(fold-change)
I
P-value
P-value
H
P-value
G
Log2(fold-change)
Log2(ratio)
F
E
Log2(fold-change)
EPF vs CPF
Log2(ratio)
Log2(fold-change)
D
P-value
EPF vs 0PF
Log2(fold-change)
C
P-value
B
Ectopic cells
P-value
Log2(ratio)
P-value
A
P-value
CPF vs 0PF
Control cells
Log2(ratio)
Log2(fold-change)
Results
V u l c a n o
Plots
cultures
The major difference in miRNAs expression was observed in eutopic cells after EPF
treatment
Eutopic cells
Log2(fold-change)
P-value
Log2(fold-change)
Log2(fold-change)
I
P-value
P-value
H
P-value
G
Log2(fold-change)
Log2(ratio)
F
E
Log2(fold-change)
EPF vs CPF
Log2(ratio)
Log2(fold-change)
D
P-value
EPF vs 0PF
Log2(fold-change)
C
P-value
B
Ectopic cells
P-value
Log2(ratio)
P-value
A
P-value
CPF vs 0PF
Control cells
Log2(ratio)
Log2(fold-change)
Results
V e n
Diagrams
culturesControl cells
Eutopic cells
Ectopic cells
CPF vs 0PF
CPF vs 0PF
CPF vs 0PF
267
9
6
1
0
9
EPF vs 0PF
0
2
EPF vs CPF
18
10
8
201
EPF vs 0PF
n
1
8
0
0
0
12
13
1
58
EPF vs CPF
2
EPF vs 0PF
72
82 mature miRNAs differently expressed (EPF vs 0PF)miRNAs
10
miRNAs
EPF vs CPF
Results
miRNA selected for
validation miR-185-5p
miR-149-5p
qRT-PCR validation
Endogenous control: RNA RNU6B
miR-130a-5p
miR-21-5p miR-106b-5p
2
*
1
0PF CPF EPF
0PF CPF EPF
12
10
8
6
2
0
0PF CPF EPF
**
4
0PF CPF EPF
14
7
12
6
5
4
**
2
1
0
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
*
4
2
0PF CPF EPF
8
3
6
0
0PF CPF EPF
miR-195-5p
(Relative expression)
0
12
miR-106b-5p
miR-21-5p
3
Proteolysis
miR-16-5p
Control
10endometrium
miR-29c-3p
Eutopic endometrium
8 endometrioma
miR-424-5p
Ovarian
14
miR-185-5p
miR-130a-5p
4
miR-195-5p
Angiogene
sis
0PF CPF EPF
0PF CPF EPF
10
8
6
4
*
2
0
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
Results
miRNA selected for
miR-149-5p
qRT-PCR validation
miR-130a-5p
2
*
1
0PF CPF EPF
0PF CPF EPF
12
10
8
6
2
0
0PF CPF EPF
**
4
0PF CPF EPF
14
7
12
6
5
4
**
2
1
0
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
*
4
2
0PF CPF EPF
8
3
6
0
0PF CPF EPF
miR-195-5p
0
12
miR-106b-5p
miR-21-5p
3
Proteolysis
miR-16-5p
Control
10endometrium
miR-29c-3p
Eutopic endometrium
8 endometrioma
miR-424-5p
Ovarian
14
miR-185-5p
miR-130a-5p
4
miR-195-5p
Angiogene
sis
0PF CPF EPF
0PF CPF EPF
10
8
6
4
*
2
0
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
Results
miRNA selected for
miR-149-5p
qRT-PCR validation
miR-130a-5p
2
*
1
0PF CPF EPF
0PF CPF EPF
12
10
8
6
2
0
0PF CPF EPF
**
4
0PF CPF EPF
14
7
12
6
5
4
**
2
1
0
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
*
4
2
0PF CPF EPF
8
3
6
0
0PF CPF EPF
miR-195-5p
0
12
miR-106b-5p
miR-21-5p
3
Proteolysis
miR-16-5p
Control
10endometrium
miR-29c-3p
Eutopic endometrium
8 endometrioma
miR-424-5p
Ovarian
14
miR-185-5p
miR-130a-5p
4
miR-195-5p
Angiogene
sis
0PF CPF EPF
0PF CPF EPF
10
8
6
4
*
2
0
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
Results
miRNA selected for
miR-149-5p
qRT-PCR validation
miR-130a-5p
1
*
*
**
0
0PF CPF EPF
*
0PF CPF EPF
*
10
6
4
**
2
0PF CPF EPF
4
3
2
*
1
0
0PF CPF EPF
0PF CPF EPF
Control en
Eutopic en
Ovarian en
8
0
0PF CPF EPF
Proteolysis
miR-16-5p
miR-29c-3p
miR-424-5p
12
miR-29c-3p
Control endometrium
Eutopic endometrium
Ovarian endometrioma
miR-424-5p
miR-16-5p
2
miR-195-5p
Angiogene
sis
0PF CPF EPF
0PF CPF EPF
0PF CPF EPF
Results
Results
F u n c t i o n a l
experiments:
!  miR-16-5p mimic
!  miR-29c-3p mimic
Transfected
Control
cells
Regulatory effect
on VEGF-A
expression.
Eutopic
cells
EAHY 926
VEGF-A protein levels [pg/ml]
Dose-dependent reduction of VEGF-A expression after mimic transfection
125
20 nM
50 nM
100 nM
100
75
50
25
0
Scr
-16-5p
-29c-3p
-424-5p
miRNA
The reduction of VEGF-A protein levels without a concomitant reduction in VEGFA
mRNA expression suggests the involvement of a postranscriptional mechanism of
regulation.
Results
F u n c t i o n a l
experiments:
!  miR-29c-3p mimic
Transfected
Control
cells
Regulatory effect
on VEGF-A
expression.
Eutopic
cells
EAHY 926
125
125
100nM
100
75
50
25
0
VEGF-A mRNA
(relative to scramble)
VEGF-A protein levels [pg/ml]
Dose-dependent reduction of VEGF-A expression after mimic transfection
75
50
25
0
Scr
-16-5p
-29c-3p
miRNA
-424-5p
Scramble
miR-16-5p
miR-29c-3p
miR-424-5p
100
Scramble
miR-16
miR-29c
miR-424
Cell culture
The reduction of VEGF-A protein levels without a concomitant reduction in VEGFA
mRNA expression suggests the involvement of a postranscriptional mechanism of
regulation.
Results
F u n c t i o n a l
experiments:
Reduction in VEGF-A expression after mimic transfections
(100nM)
Scr
-16-5p
-29c-3p
Scr
EAHY 926:
Scr
-16-5p
-29c-3p
-424-5p
Control cells:
Scr
Eutopic cells:
-16-5p
-29c-3p
-424-5p
-424-5p
Results
Luciferase assays:
Internal control: Renilla
luciferase
pMIR-REPORT Firefly Luciferase
+ 3’ UTR VEGF-A
Co-Transfection with
miR-29c-3p or scramble mimics
HTC116-DICER
KO
Cell line
24h
Assay luciferase activity
pMIR-REPORT Firefly Lucifera
+ 3’ UTR VEGF-A MUT
(Deleted miR-29c-3p binding site)
Results
Luciferase assays:
luciferase
+ 3’ UTR VEGF-A
HTC116-DICER
KO
Cell line
24h
Results
Luciferase assays:
luciferase
+ 3’ UTR VEGF-A
HTC116-DICER
KO
Cell line
24h
Results
(Luciferase/Renilla) ratio
Luciferase assays:
150
Scramble
100
miR-29c-3p
***
50
0
WT
Mut
Luciferase activity was lower when the vector containing the 3’UTR wild type was
cotransfected with miR-29c-3p in comparison to scramble mimic.
Results
(Luciferase/Renilla) ratio
Luciferase assays:
150
Scramble
100
miR-29c-3p
***
50
0
WT
Mut
Luciferase activity was lower when the vector containing the 3’UTR wild type was
cotransfected with miR-29c-3p.
In contrast, when the binding site for miR-29c-3p was deleted, the transfection
with mimic for miR-29c-3p showed similar results to scramble.
Conclusion
s
1.  This “in vitro” study indicates that peritoneal fluid from women with
endometriosis modulates the expression of miRNAs that could
contribute to the angiogenic disequilibrium observed in this disease.
2.  Array results revealed that both peritoneal and endometrial factors
contribute to the observed disequilibrium in miRNA expression.
3.  These results point to a contribution of both factors in the
pathophysiology of endometriosis.
4.  Luciferase assays ensure that miR-29c-3p regulates VEGF-A
expression.
Thank you for your
attention!

Peritoneal fluid modifies the miRNA expression profile in

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