Micro RNAs: potentielle Biomarker für das. Blutspenderscreening

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1 Micro RNAs: potentielle Biomarker für das Blutspenderscreening

2 micrornas - Background Types of RNA -Coding: messenger RNA (mrna) -Non-coding (examples): Ribosomal RNA (rrna) Transfer RNA (trna) Small nuclear RNA (snrna) Small nucleolar RNA (snorna) Short interfering RNA (sirna) Micro RNA (mirna)

3 micrornas - Background Short, single-stranded, endogenous, non-coding RNAs Found in animals, plants, Originate from capped & polyadenylated full length precursors (pri-mirna) Hairpin precursor ~70 nt (pre-mirna) Mature mirna ~22 nt (mirna) First discovered in 1993 by Victor Ambros at Harvard (lin-4) Now: thousands of microrna molecules:

4 mirna statistics (2010) Total mirna genes in 115 species 10,882 Total number of mirnas known 1,580 Number human mirnas identified 851 Number of human mrna targets 34,788 Each mirna appears to regulate the translation of multiple genes, and many genes appear to be regulated by multiple mirnas

5 The mirna processing pathway - a primary mirna (pri-mirna) transcript is encoded in the cell's DNA and transcribed in the nucleus, - processed by an enzyme Dosha - and exported into the cytoplasm where it is further processed by Dicer. - after strand separation - the mature mirna represses protein production either by blocking translation or causing transcript degradation. Many roads to maturity: microrna biogenesis pathways and their regulation Julia Winter, Stephanie Jung, Sarina Keller, Richard I. Gregory & Sven Diederichs Nature Cell Biology 11, (2009)

6 micrornas -Function mirna regulates post-transcriptional gene expression by binding to complementary sequences in the target gene Perfect complementarity of binding leads to mrna degradation of the target gene Imperfect pairing inhibits translation of mrna to protein Important in development: embryogenesis and differentiation, regulate cell proliferation, apoptosis Metabolic regulation (mir-375 & insulin secretion)

7 MicroRNAs Commonly Associated with Human Cancer mirnas control cell cycle, cell differentiation and apoptosis by regulating oncogenes and tumor supressor genes mirnas are misexpressed in cancer and are therefore excellent diagnostic/prognostic markers in cancer Some mirnas e.g. mir-155, can cause cancer and oncogenic mirnas may be therapeutic targets in cancer Other mirnas like let-7, may prevent cancer and may be therapeutic molecules themselves. MicroRNAs could augment current cancer therapies.

8 Role of mirnas in Host-Virus Interaction Circumstantial evidence suggests that mirna play a role in the mammalian virus-host interactions by limiting virus replication and viruses may impede RNA silencing pathways as a defense to these attacks. Lecellier, C.H., et. al. A Cellular microrna Mediates Antiviral Defense in Human Cells. Science. 2005; 308:

9 microrna as diagnostic marker Emerging role of micrornas in liver diseases Shashi Bala, Miguel Marcos, Gyongyi Szabo World J Gastroenterol December 7, 2009 Volume 15 Number 45

10 Study Circulating plasma mir-122, mir-155, and mir-16 (HCV-induced), and mir-122, mir-146a, and mir-181a (HBV-induced) were measured by quantitative PCR in seroconversion panels. Synthetic Caenorhabditis elegans mir-238 was spikedin as internal control and for normalization. mirna expression patterns were compared based on cycle threshold values.

11 Reverse Transcription with looped-primer: - stem-loop increase melting temperature - the last 6 nt are specific to the mirna mirna-specific TaqMan MGB probe with nonfluorescent quencher (NFQ)

12

13

14 Conclusions mir-155, -16 (HCV), -146a and 181a have no potential to be used as markers for acute HCV- or HBV-infections Host derived micrornas are less specific than classical infection parameters and may only serve as additional markers for blood donor screening as shown in case of mir-122: - mir-122 is physiologically expressed in healthy subjects; - a test would have to be quantitative to distinguish between infected and non-infected individuals - mir-122 expression is also influenced by liver injury caused by various pathogens or intoxication, resulting in individual differences in mir-122 levels.

15 Vielen Dank für Ihre Aufmerksamkeit!

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