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: mgood@kids.wustl.edu