MicroRNAs: a new source of biomarkers in radiation response. Simone Moertl, Helmholtz Centre Munich
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1 MicroRNAs: a new source of biomarkers in radiation response Simone Moertl, Helmholtz Centre Munich
2 The RNA World mrna rrna coding snorna trna scarna RNA snrna noncoding lincrna rasirna RNAi anti-sense pirna mirna sirna
3 microrna - characteristics nt RNA molecules ~2000 human mirnas known encoded in the nucleus several processing steps to produce mature mirna negative regulators of gene expression one mirna can target several mrnas and one mrna can be targeted by several mirnas about 40 % of all genes are regulated by mirnas
4 mirna biogenesis and function regulation activation/repression of transcription (p53, myc, promotor methylation) regulation of maturation mirna stability
5 mirna biogenesis is induced by DNA damage through ATM ATM pri-mirna pre-mirna Zhang et al., Mol Cell Feb 18;41(4):
6 fold change relative metabolic activity global mirna knockdown radiosensitizes cells mir-10b mir-518b mir-101 mir 523-5p sinegative siago2 sidicer sinegative siago2 sidicer dose [Gy] Kraemer and Moertl, RAD.RES 2011
7 mir-525-3p regulates cellular radiation sensitivity in endothelial cells mir-525-3p control mir-525-3p overexpr. mir-525-3p anti-mir Kraemer and Moertl, PlosOne (2013)
8 Validation of differentially expressed proteins after inhibition of mir-525-3p Kraemer and Moertl, PlosOne (2013)
9 Radiation response after depletion of ARRB1 and TXN1 survival TXN1 mir-525-5p ARRB1 Kraemer and Moertl, PlosOne (2013)
10 mirnas as biomarker for individual radiosensitivity (RADSENS) RTGO 0 RTGO 3? pathways triggered by low dose radiation? pathways differing in sensitive/non sensitive patients mirnome analysis:1152 mirnas in vitro irradiation (whole blood) 1 mgy 150 mgy sample RTGO 3/ RTGO 0 RTGO 3, 150 mgy 20 down, 11 up 23 down, 24 up RTGO 0, 150 mgy 18 down, 15 up mirna expression (lymphocytes) RTGO 3, 1 mgy RTGO 0, 1 mgy 61 up 41 down, 24 up
11 mirna expression changes after low dose irradiation
12 CT values CT values CT values CT values mirnas are differentially expressed in RTGO 0 versus RTGO 3 patients mir-let-7f mir * * RTOG 0 RTOG 3 RTOG 0 mir-486-5p RTOG RTOG 0 RTOG 3 RTOG 0 mir-490-3p RTOG 3 * * RTOG 0 RTOG 3 RTOG 0 RTOG 3 * p< 0,05 RTOG 0 RTOG 3 0 RTOG 0 RTOG 3
13 mirna expression discriminates blood samples from RTGO 0 and RTGO 3 patients mirna sensiti vity specifici ty let-7c 0,778 1,00 mir-19a 0,667 1,00 mir-381 0,889 1,00 mir-486 0,667 0,89 mir-490 0,889 1,00 mir-514a 0,556 0,67
14 Extracellular mirnas (Turchinovich, Weiz et al. 2012) 14
15 # of mirnas detected Circulating mirnas as biomarker during radiotherapy Healthy donors Head and neck cancer patients p= plasma samples before and during radiochemotherapy Quantitative microrna-profiling in blood plasma (RealTime-PCR) mirnas as: patients (non-irr.) 33 healthy donors diagnostic or prognostic tools radiotherapy monitoring Summerer et al., BJC (2015)
16 Circulating mirna expression in healthy donors and HNSSC patients Summerer et al., BJC (2015)
17 Circulating mirnas expression before and after radiotherapy Summerer et al., Radiation Oncology (2013)
18 High expression of mir-142-3p post treatment indicates poor locoregional tumor control discovery cohort, n = 15 validation cohort, n = 11 Summerer et al., BJC (2015)
19 Exosome mirnas as messages in intercellular communication paracrine endocrine exosomes protect RNA by their lipid bilayer contain RNA, proteins and lipids may stimulate surface receptors acting in neighboring cells or distant tissues Onco Targets Ther Jul 21;7:
20 Exosomal mirnas as biomarker for radiation whole blood in vitro irradiation exo. isolation characterization mirna expression
21 Exosome tracking analysis
22 Time dependent uptake of exosomes Exo 3h h 6 h 24 h 6h 24h
23 n-fold change mirna expression in exosomes mirnas up 3 mirnas down mir-204-5p 0 mir-124-3p mir-7-5p mir-145-5p mir-143-3p mir-130b-3p mir-423-3p mir-224-5p mir-21-5p mir-18a-5p mir-24-3p mir-192-5p mir-20a-5p mir-221-3p mir-126-3p mir-122-5p mir-31-5p
24 n-fold change Donor 1 Donor 2 Donor 3 Donor 4 Donor 5 Donor 6 Radiation changes exosomal mirna expression 2 1,5 * * * * 1 0, fold 0.25-fold 0 Control mir-31-5p mir-92a-3p mir-204-5p
25 Summary cellular mirnas cellular mirna expression is triggered by radiation cellular mirna influences radiation sensitivity PBMNC mirna expression might predict individual sensitivity circulating RNA Biomarker for HNSSC respondes to radiation potential as therapy marker exosomal mirnas radiation changes exosomal mirna loading
26 mirna : target recognition seed sequences partial duplex mrna : mirna mostly 3 - UTR interactions seed sequences (nt 2-7) centered sites (nt 12-17) 3 - interactions nucleotide bulges (1-5 nt loops) Pasquinelli A., Nature Reviews Genetics, 2012
27 High expression of mir-574-3p indicates poor locoregional tumor control discovery cohort, n = 15 validation cohort, n = 11 Summerer I, unpublished
28 Exosomes Intercellular communication direct cell-to-cell contact messenger substances hormones and exosomes Features: nm diameter bilayer lipid membrane store proteins and RNA-molecules Biogenesis of exosomes clathrin coated pits early endosome multivesicular endosome fusion with the cell membrane results in the release of exosomes Biogenesis of exosomes (Raposo and Stoorvogel 2013) 28
29 Electron microscopy image of exosomes Electron microscopy of exosomes from human plasma. [scale: 100 nm] 100 nm Size and morphology corresponds to the features of exosomes 29
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