mi micr cro-RNAs RNAs as bio s bioma marker rkers s in in childr chi ldren en wh who und o underwent rwent sur surgery gery for for CHD CHD ment entor ors: s: Dr. . Yael el Ne Nevo vo - Casp Caspi i & Prof. of. Gid idi Paret aret Or Berc rcovich ovich 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 ork • Wh What at ’ s s next xt
Congenital heart defects (CHD) • Inci cidenc dence: : 8/1000 1000 • Most t co comm mmon on 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 ostl tly y spor oradic adic muta tati tions • Attitudes tudes towar ard d pregnan nancy cy termi mination nation • Cya yanotic otic vs. non-cy cyanoti anotic
Congenital heart defects (CHD) • Treatment: ment: • Some ome defec ects s do not ot need eed interven erventi tion • Medic icat atio ions (diur iureti etics, cs, digoxin oxin, , 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 ortal 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 ogical al and d renal nal compli omplicat ation ions
The need for early diagnosis • Early ly dia iagn gnosis sis of compli lica cation tions s may im improve ove 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 oponin 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 oRNA 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 ome (over er a t thousa usand nd have ve been iden entifi tified ed in human an) • Main role: post-tra transc nscript riptional onal regulati lation on • 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 ory 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 otential 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 No micro-RNA-based drug is in the 3. . Bio io- av avai aila lability bility market … yet 4. . Cost t eff ffective ectivene ness ss
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 ology 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 ood Bonus: may predict the 5. Relia liable ble testing sting prognosis 6. Simp imple, e, quick uick and d reproducibl eproducible e diagnosi iagnosis
Mentors: Prof. Gidi Paret Dr. Yael Nevo-Caspi Or Bercovich Pediatric intensive care unit – Sheba Medical Center 15
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 o und nderwe erwent nt surger rgery y for CHD Inclu cluded ed cases ses Exclu luded ed cases ses N= N=75 75 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 onse 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 onse 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 omote inflamm nflammator atory y me mediat diator ors ◦ Important ortant marker ker of immun mune e cell ll acti tivati vation on 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 on 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 ore surgery rgery ◦ 6h ◦ 12 12h ◦ 24 24h RQ calcul ulati ation: on: ◦ 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
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Continu inue e lab work Statisti istical cal anal alysis ysis Writing ng 27
Immun unom omod odulat 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 on 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)- Scale of 1-6 cognitive function following a critical illness or injury Pediatric Stroke Outcome Measure (PSOM)- Scale of 5 subscales: right+ left sensorimotor, language production+ 0-2 per comprehension, and cognitive function function Disadvantages: - Gross assessment based on observer ’ s impression - Insignificant while child is unstable late diagnosis - Do not indicate prognosis
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 Post-operation neurological damage neurological damage N=20 N=20
Deciding on miRs was difficult … Requirements Difficulties Brain specific/ 70% of known miRs are brain enriched miR expressed in the brain Some promising miR are Present in serum un-detectable in serum Dysregulation of Inconsistent trends of most expression due to miRs in different studies neurological damage
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