The Role of MicroRNAs in NEC · Betel et al, Nucleic Acids Res 2008 Good et al, Mucosal Immunology,...
Transcript of The Role of MicroRNAs in NEC · Betel et al, Nucleic Acids Res 2008 Good et al, Mucosal Immunology,...
The Role of MicroRNAs in NEC
Misty Good, MDNeonatologist, Assistant Professor of Pediatrics
Division of Newborn Medicine, Children’s Hospital of PittsburghDepartment of Pediatrics, University of Pittsburgh School of Medicine
September 26, 2016
Objectives
• Review the pathogenesis of NEC.• Describe the mechanism by which breast
milk is protective against NEC. • Discuss the role of microRNAs in NEC.
Introduction• Necrotizing enterocolitis is the leading cause of death from
GI disease in the premature infant• Etiology remains incompletely understood
Premature infant with NEC Intraoperative
Hackam et al. Clin Dev Immunol. 2013.
How does NEC happen?
Hypoxia
Background
• TLR4 activation within intestinal epithelium leads to mucosal injury– Through accelerated enterocyte apoptosis– Impaired mucosal proliferation
• Since TLR4 is important in NEC development, strategies to limit TLR4 signaling may offer a preventative approach
Background
• NEC is up to 6x more common in infants fed formula vs. breast milk
• The specific protective agent and mechanism mediating protection is unclear
Background
• EGF is involved in intestinal development• present in breast milk and amniotic fluid
• EGF enhances proliferation of epithelial cells and heals damaged mucosa
• EGF is involved in regulation of cell replication, cell movement and cell survival
Harris et al, EGF receptor ligands. Exp Cell Res 2003;284:2-13.
Duh et al. EGF regulates early embryonic mouse development in chemically defined organ culture. Pediatr Res 2000;48:794-802
Hypothesis
• We hypothesized that breast milk inhibits TLR4 signaling within the neonatal intestinal epithelium via EGFR activation, and attenuates the severity of experimental NEC.
Induction of mouse experimental NECNeonatal mouse pups
Breast fed controls
Gavage NEC formulaevery 3 hours for 12hrs
Hypoxia (5% O2, 95% N2)twice daily
NEC in 4days
Free air
Decreased intestinal EGFR expression is associated with NEC development in mice
Good et al, PNAS, 2012BF NEC
Decreased intestinal EGFR expression is associated with NEC development in humans
Good et al, PNAS, 2012
Breast Milk Extraction
Breast milk inhibits TLR4 signaling in vivoControl LPS LPS+BM LPS+BM+Cetux
LPS
v
Saline LPSBM
LPSBM
Cetux
Tota
l Flu
x (p
hoto
ns/s
ec) x
104
Nor
mal
ized
to c
ontro
l
8
0
*
**
***
ii iiii iv
Saline LPSBM
LPSBM
CetuxTL
R4
qRT-
PC
R
5
0LPS
*
**
***
vii
Saline LPSBM
LPSBM
Cetux
IL-6
qR
T-P
CR
22
0LPS
*
**
***
vi
Good et al, Mucosal Immunology,
2015
Breast milk attenuates NEC via EGFR activationWild-type
i ii iii
iv v vi NEC+IBM+EGF
Control NEC NEC+BM
NEC+IBM NEC+BM+Cetux
ii
NEC NEC BM
NEC IBM EGF
Ctrl
*
**
NEC BM
Cetux
*** ***
NEC
Sev
erity
Sco
re
3
0 NEC IBM
****
i
NEC NEC BM
NEC IBM EGF
Ctrl
*
**
NEC BM
Cetux
***
***
iNO
S q
RT-
PCR
30
0 NEC IBM
**** Good et al, Mucosal Immunology,
2015
Breast milk attenuates NEC severity via EGFR
EGFR∆IEC
Control NEC NEC+BM i ii iii
NEC Ctrl
NE
C S
ever
ity S
core
3
0
*
NEC BM
ii *
8
NEC Ctrl
iNO
S q
RT-
PC
R
0
*
NEC BM
i
*
Good et al, Mucosal Immunology,
2015
Breast milk inhibits NEC-mediated apoptosis and enhances crypt proliferation via EGF
A
TUNEL3NTDapi
i ii iii
iv v vi
Wild-typeControl NEC
NEC+IBM
NEC+BM+
Cetux
Control NEC
NEC+IBM
NEC+BM+
Cetux
PCNADapi
i ii iii
iv v vi
TUNEL3NTDapi
PCNADapi
B
NEC+BM
NEC+IBM+EGF
C*** i
*
Wild
-type
TUN
EL+
ce
lls/v
illus
NEC NECBM
NECIBMEGF
Ctrl
**
NECBM
Cetux
***8
0 NECIBM
****
Wild
-type
PC
NA
+ c
ells
/cry
pt 12
NECIBMEGF
NECBM
Cetux
NEC NECBM
Ctrl
*
**
*** ***
0 NECIBM
****iiNEC+
BM
NEC+IBM+EGF Good et al,
Mucosal Immunology, 2015
Breast milk inhibits NEC-mediated apoptosis and enhances crypt proliferation via EGFR
EGFR∆IEC
TUNEL3NTDapi
Control NEC NEC+BM
Control NEC NEC+BM
i ii iii
i ii iii
PCNADapi
C
D
NECCtrl
EG
FR∆
IEC
PC
NA
+ c
ells
/cry
pt
12
0
*
NECBM
ii
*NEC NEC
BMCtrl
* *
EG
FR∆
IEC
TUN
EL+
cel
ls/v
illus 8
0
iE
Good et al, Mucosal Immunology,
2015
Breast milk inhibits TLR4 signaling in intestinal epithelial cells via EGFR activation
ii iii
NF-κB
i ivControl LPS LPS+BM
IEC
-6
LPS+IBMv
LPS+IBM+EGF
EGFR
-kd
LPS+BBMvi
ii iiii iv v
NF-κB
Wild-type IEC-6
LPSCtrl
IL-6
qR
T-P
CR
3
0
*
**
BMLPSBM
iWild-type IEC-6
ii
TLR
4 qR
T-P
CR
4
LPSCtrl LPSBM
BM0
*
**
iiiEGFR-kd IEC-6
LPS LPSBM
BMCtrl
*5
0IL
-6 q
RT-
PC
R
*
Good et al, Mucosal Immunology,
2015
Interim Summary
• We have shown that breast milk is protective against NEC by inhibiting TLR4 with the activation of EGFR.
• EGFR signaling protected against TLR4-mediated enterocyte apoptosis and enhanced enterocyte proliferation in NEC.
• Thus, it is particularly important to study the gene regulation of EGFR in NEC as a therapeutic or preventative target in NEC.
MicroRNA Background
• Noncoding RNA about 22 nucleotides long
• Functions in RNA silencing and therefore post-transcriptional regulation of gene expression
• Initially expressed as pri-miRNAs in clusters that undergo post-transcriptional processing to produce mature miRNAs
miRNA Background
• MiRNAs are differentially expressed in the intestine.
• Implicated in IBD, intestinal inflammation, gut barrier function.
Observation:
miRNAs are known to be differentially expressed in intestinal inflammatory states
Hypothesis:miRNAs are differentially expressed in premature
infants with NEC
• Univ. of Pittsburgh approved IRB protocol
• Inclusion/Exclusion Criteria• Premature infants admitted to NICU• NEC patients with Bell’s Stage II or greater• Excluded patients with congenital anomalies
• Samples• Intestinal resections: At NEC diagnosis or at stoma closure
(Resolved NEC)• Serum: At time of NEC diagnosis or age-matched controls
Methods
NEC is Associated with Differential Expression of miRNAs
miRNA-17~92 Background
• MiR17~92 cluster made up of miRNA-17, 18a, 19a, 19b, 20a, and 92a
• These miRNAS are known to be differentially expressed in intestinal inflammatory states.
• Components of the miR-17~92 cluster have increased intestinal and serum expression in inflammatory states of IBD.
Embryonic small intestine of miR-17~92ko mice display blunted villiform structures
Control miR-17~92ko
• Previously our lab has shown that EGFR expression is decreased in NEC
• Protective effects seen in NEC with EGFR activation
• miRNA-17 is predicted to target EGFR
Good et al, PNAS, 2012Betel et al, Nucleic Acids Res 2008Good et al, Mucosal Immunology, 2015
MicroRNAs
Expression of Intestinal miRNA-17 Increased in NEC
0
2
4
6
8
miR
-17
qPC
R e
xpre
ssio
n
**
**P<0.002
Pompa et al, in preparation
Serum miRNA-17 is Increased in NEC
0
5000
10000
15000
20000
25000
seru
m m
iR-1
7 qP
CR
*
*P<0.05
Pompa et al, in preparation
Serum MiR-17 is increased in medical and surgical NEC
Control
Medica
l NEC
Surgica
l NEC
0
20
40
60
Rel
ativ
e m
iRN
A-1
7 Ex
pres
sion
*
*
*P<0.05
Pompa et al, in preparation
Observation:
miRNA-17 is increased in the intestine and blood of infants with NEC
Hypothesis:
Infants with increased miRNA-17 have increased intestinal barrier dysfunction and decreased cellular proliferation
miRNA-17 expression is increased in surgical NEC
miR-17 LNA-ISH
NEC Resolved NEC
Pompa et al, in preparation
Increased miR-17, decreased proliferation, tight junctions and EGFR in NEC
NEC Resolved NEC
ZO-1PCNADAPI
EGFR
Pompa et al, in preparation
MiR-17 targets EGFR
Egfr +
Tomato
Egfr +
miR17
0
50
100
150
Rel
ativ
e Lu
cife
rase
(%)
*
Pompa et al, in preparation
Conclusions
• The miRNA-17~92 cluster is upregulated in the intestines of infants with NEC
• miRNA-17 is upregulated in the intestine and serum of infants with medical and surgical NEC.
• MiR-17 targets EGFR• miRNA-17 may play an important role in the
maintenance of the gut barrier in NEC pathogenesis
Future Directions
• Characterize the intestinal cell populations that express miRNAs with flow cytometry.
• Evaluate susceptibility of intestinal specific miRNA-17~92ko mice NEC model.
• Determine the effects of Mir-17~92 cluster on intestinal development in the mouse.
Future Directions
• Determine the effect of overexpressing Mir-17 in neonatal mouse enteroids with and without NEC.
Neonatal mouse enteroids
EcadMuc2
LysozymeDAPI
Funding SourcesNational Institutes of Health K08DK101608CHP Department of Pediatrics UPMC Competitive Medical Research Fund
David Hackam Lab, Hopkins
Acknowledgements
Chhinder Sodhi, PhDHongpeng Jia, MDCharlotte Egan, PhDYuki Yamaguchi, PhDPeng Lu, PhDAmin Afrazi, MD, PhDMaria BrancaTom PrindleSamantha Weyandt
Jay Kolls Lab, PittPawan Kumar, PhD
Kara KracinovskyGary Silverman Lab, Wash U
Cliff Luke, PhD
John Ozolek, MD, Pitt
Good LabAnthony Pompa, MD (Pediatric Resident)Congrong Ma (Technician)Alexa Bolock (Technician)Zerina Hodzic (Medical Student)Olivia Parks (Undergraduate Student)Kara Touscany (Undergraduate Student)Onome Oghifobibi, MD (Pediatric Resident)Sonali Agrawal (Undergraduate Student)Tyler McCullough (Undergraduate Student)
Jackie Ho Lab, PittYu Leng Phua
Thank you! Questions?
The Good Lab is relocating to Washington University in St. Louis, MO, USA and is hiring…
if interested please email: [email protected]