mi micr cro-RNAs RNAs as bio s bioma marker rkers s in in - - PowerPoint PPT Presentation

mi micr cro-RNAs RNAs as bio s bioma marker rkers s in in chi childr ldren en wh who und

• underwent

rwent sur surgery gery for for CHD CHD

ment entor

• rs:

s: Dr. . Yael el Ne Nevo vo-Casp Caspi i & Prof.

• f. Gid

idi Paret aret Or Berc rcovich

• vich

Liat at Mor

What will we talk about…

• Congenital

ngenital hear art t defects fects (CHD) HD)

• Scientific

ientific bac ackground kground

• mi

miRNAs NAs in CHD HD

• La

Lab work

• rk
• Wh

What at’s s next xt

Congenital heart defects (CHD)

• Inci

cidenc dence: : 8/1000 1000

• Most

t co comm mmon

• n birth

th defect ct

• Causes

es:

• Unkno

known

• Infecti

fections (e.g.

• g. rubell

ella) a)

• Medic

icat atio ion (e.g. . Thalido halidomide) ide)

• Alcoh

cohol/To l/Tobac bacco co

• Inbr

bree eedin ing

• Nu

Nutritio tritional l sta tatus us (under ndernu nutrit itio ion, DM, etc tc)

• Gene

netic ic – mos

• stl

tly y spor

• radic

adic muta tati tions

• Attitudes

tudes towar ard d pregnan nancy cy termi mination nation

• Cya

yanotic

• tic vs. non-cy

cyanoti anotic

Congenital heart defects (CHD)

• Treatment:

ment:

• Some
• me defec

ects s do not

• t need

eed interven erventi tion

• Medic

icat atio ions (diur iureti etics, cs, digoxin

• xin,

, etc)

• Cathet

theter er based ed proce cedures dures

• Heart

art surgery gery

• Hear

art transplan nsplant

• Compli

plica catio tions ns after r surger gery

• Leadin

ading g cause use of birth th defect ect related ated death ths

• Early

ly mor

• rtal

alit ity – 5-10 10%

• SIRS

RS (1/3 3 of cases) es)

• Arrhy

hyth thmi mias as and nd heart eart failu lure re

• Lun

ung g injury jury

• Ne

Neurol urologic

• gical

al and d renal nal compli

• mplicat

ation ions

The need for early diagnosis

• Early

ly dia iagn gnosis sis of compli lica cation tions s may im improve

• ve treatmen

ment t and it its outco comes mes

• Today’s bio

iomar arker ers: s:

• Bioma

markers rkers for myo yocard cardial al injury: ry:

• Tropon
• ponin

in, CPK, BNP

• Cardiac

diac miRNA RNA

• Th

Ther ere e ar are e no spec ecifi ific c inflamma lammatory tory biomarkers markers

MicroRNA

• Micr

croR

• RNA

NAs s (miRNA NAs) s) are small ll non-codi coding ng RNA molec ecul ules es

• They

y co consi sist st 19 19-24 24 nucl cleoti eotides des

• Consti

stitute tute 1-3% of the huma man n genome

• me (over

er a t thousa usand nd have ve been iden entifi tified ed in human an)

• Main role: post-tra

transc nscript riptional

• nal regulati

lation

• n
• Inh

nhibi ibit mRNA RNA translat nslatio ion

• Promo

mote e mRNA degrada adatio ion

MicroRNA

• Ov

Over r 50 50% of huma man n genes s are likel ely y regula lated ted by y miRNA NA

• Ti

Tissue-spec specific fic ex expre ression ssion pat atte tern rn

• Dys

ysregulat egulated ed miRNA A expre ression: ssion:

• Canc

ancer er

• Infl

flam ammat atory

• ry diseases

eases

• Auto

toim immu mune e diseases eases

• Role in immune

mune sys ystem: em:

• Immu

mmunom nomodu dulat latio ion n and nd fine ne-tu tunin ning

MicroRNA

• Circulating miRNAs in the serum:
• Cell damage and cell death
• Cell communication
• High stability
• Ribonucleoprotein complex
• Intercellular communication via extracellular vesicles (EVs)
• Ectosomes
• Exosomes
• Apoptotic bodies
• Also found in urine, saliva, CSF and breast milk
• Breast milk miRNAs may have a role in immunoregulation

MicroRNA - biogenesis

mRNA degradation mRNA cleavage

Repression of gene expression

A promising field of research…

• Pot
• tential

ntial th therapeutic apeutic us use

Requirements: quirements: 1. . Specificity ecificity of f mi miR to pat athology hology 2. . No/minimal /minimal side e eff ffects ects 3. . Bio io- av avai aila lability bility 4. . Cost t eff ffective ectivene ness ss

No micro-RNA-based drug is in the market…yet

A promising field of research…

• Specific

cific bio iomarke markers rs

Requi quirem emen ents: ts:

• 1. Specifi

ecificity city of miR to p path tholo

• logy

gy

• 2. Signi

gnifican ficant t change ange in express ression ion

• 3. Circul

rculatin ating g miRs Rs

• 4. Stab

able le in blood

• od
• 5. Relia

liable ble testing sting

• 6. Simp

imple, e, quick uick and d reproducibl eproducible e diagnosi iagnosis

Bonus: may predict the prognosis

Mentors: Prof. Gidi Paret

• Dr. Yael Nevo-Caspi

15

Or Bercovich

Pediatric intensive care unit – Sheba Medical Center

 Examination of the association between the expression of

immunomodulatory miRNAs and the inflammatory response following surgery for CHD

 Development of a diagnostic tool that will improve medical

management and outcome following surgery for CHD

16

Chil ildren ren who

• und

nderwe erwent nt surger rgery y for CHD Inclu cluded ed cases ses N= N=75 75 Exclu luded ed cases ses N = 4 Pre-sur urgery ery infectio fections ns Immun mune disea seases es

17

 A better

ter under erstan standing ing of the inflamm ammatory atory resp sponse

• nse to

cardiac iac surgery rgery may be the key to deve velo lopment ment of successful essful strategies ategies to minimize ze patient ient morbi bidity dity and d mortal alit ity

 Infl

flamm ammatory atory resp spons

• nse

e to cardiac ac surg rger ery:

• Preven

vent t infections fections

• Wound

und healin ling g

 Validated

ated biomar arker ers s are e essen senti tial al for guiding ng drug ug therap rapy

18

 miRNA 155

155 & & 146 146a a –

• Found

und in EVs

• Re

Regul ulate ate ma many y aspect cts s of the immun mmune e respon sponse se

 miRNA 146

146a and 146 146b b –

• Low expr

pression ession may cause se a hyperactive eractive immun une response sponse

19

 miRNA 21

21 -

• high

ghly ly expr pressed essed in the fetal al heart rt

• Promote
• mote inflamm

nflammator atory y me mediat diator

• rs
• Important
• rtant marker

ker of immun mune e cell ll acti tivati vation

• n in mult

ltipl iple e contexts ntexts

21

RNA A extra raction ction from m plasma ma Prepar arat ation ion of cDNA A (Rt Rt) QRT T reaction ction Comp mpar aris ison

• n between

een levels els of miRNA RNAs and complicatio mplications ns

22

 We test

st by RQ PCR 4 4 miRNAs s for each h patien ent

 Each

h miRNA is tested ed in 4 4 differ erent ent times: s:

• 0h

h – before

• re surgery

rgery

• 6h
• 12

12h

• 24

24h

 RQ calcul

ulati ation:

• n:
• High

gh expr pression ession of a speci cific fic miRNA RNA  Fluo uores rescenc cence e in an earl rlier ier cycle cle (and d vi vice ce ve versa) rsa)

• miRNA

RNA expr pression ession after er surger urgery y of each h chil hild is compare pared d to 0h h

23

24

25

26

 Continu

inue e lab work

 Statisti

istical cal anal alysis ysis

 Writing

ng

27

 Immun

unom

• mod
• dulat

ulatory ry miRNAs s may help physic sicians ians in the future ure to take e preve venti ntive ve steps ps aga gains nst t expected ected compli licati ation

• n

28

Questions

29

Thank you!

30

MicroRNAs as Biomarkers for Brain Damage Following Cardiac Surgery in Children

Mentors:

• Dr. Yael Nevo-Caspi & Prof. Gidi Paret

Liat Mor

BACKGROUND

• Brain injury is the most prevalent post-operative

complication (40-70%)

• Wide range of neurological injuries
• Mechanism: brain hypoxia-ischemia
• Surgery induced stress and inflammation
• Weak heart due to CHD
• Cardio-pulmonary by-pass (???)

Current testing methods

Pediatric Cerebral Performance Category (PCPC)- cognitive function following a critical illness or injury Pediatric Stroke Outcome Measure (PSOM)- 5 subscales: right+ left sensorimotor, language production+ comprehension, and cognitive function Disadvantages:

• Gross assessment based on observer’s impression
• Insignificant while child is unstable late diagnosis
• Do not indicate prognosis

Scale of 1-6 Scale of 0-2 per function

Aim of study

To discover a new biomarker that will enable early diagnosis of brain damage and it’s prognosis

Why?

• 1. To allow neuroprotective therapy

(=hypothermia ???, adiponectin etc.)

• 2. To ensure close lookup on brain-functions
• 3. To adjust additional therapy and recovery plans

Research population

Children who underwent surgery for congenital heart defects

N=40

No post-operative neurological damage

N=20

Post-operation neurological damage

N=20

Difficulties Requirements

70% of known miRs are expressed in the brain Brain specific/ brain enriched miR Some promising miR are un-detectable in serum Present in serum Inconsistent trends of most miRs in different studies Dysregulation of expression due to neurological damage

Deciding on miRs was difficult…

Micro-RNA 124

• A significant brain-enriched miR
• Expressed in various neurological processes:

neurodevelopment and differentiation, neuronal degradation & stress

• The most researched brain-specific miR
• To our needs:
• Significantly elevated after 6h in stroke patients
• extent of elevation strongly correlates with

neurological outcome in rat study

Micro-RNA 107

• A brain enriched miR
• Involved in different neurological processes
• To our needs:
• Present in plasma of stroke patients
• Studies showed significant elevation of its

serum-levels within 6 hours of ischemic stroke*

Initial results

Blood samples of 3 control infants were tested for levels of miR-107 & miR-124:

• 2 had a similar trend of reduction in miRs levels
• 1 had opposite results:
• basal level was significantly higher
• the level of serum-miRs increased after surgery

His medical file revealed he had congenital hydrocephalus.

What lies ahead…

More lab work 

Better assess the clinical background and post-operative status

Statistical analysis

Find correlations!

Questions I would like to answer…

• What is the miRs trend in healthy and injured infants?
• When is the best time to withdraw blood, so that the

miR will be diagnostic?

• Is the trend of dysregulation consistent in all CHD

types?

• Are these miRs suitable biomarkers for neurological

deterioration in infants with preliminary brain damage?

• And more to come….

Thank you