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Universit Degli Studi Di Padova

Dipartimento Di Biologia

Scuola Di Dottorato Di Ricerca In Bioscienze E Biotecnologie

Indirizzo Di Genetica E Biologia Molecolare Dello Sviluppo

Ciclo XXIV

MicroRNAs: from biogenesis to post-transcriptional

regulatory networks

Direttore della Scuola: Ch.mo Prof. Giuseppe Zanotti

Supervisore: Dr.ssa Stefania Bortoluzzi

Dottorando: Marta Biasiolo

I

Contents

1. Abstract ....................................................................................................................... 1

2. Sommario .................................................................................................................... 3

3. Background ................................................................................................................. 5

3.1. miRNA biology ........................................................................................................ 5

3.1.1. microRNAs biogenesis ...................................................................................... 5

3.1.2 miRNA genes genomic arrangement .................................................................. 7

3.2. microRNA detection and their interaction with mRNAs ......................................... 9

3.2.1. MicroRNAs discovery and analysis .................................................................. 9

3.2.2. MicroRNA-mRNA interaction ........................................................................ 10

3.2.3. MicroRNA targets prediction .......................................................................... 12

3.3. miRNAs and diseases ............................................................................................ 14

3.4. MicroRNAoffset RNAs (moRNAs): by-product spectators or functional players?

...................................................................................................................................... 15

3.4.1. moRNA discovery by massive sequencing of short RNAs ............................. 16

3.4.2. moRNAs biogenesis: by-product or co-product? ............................................ 16

3.4.3. The functions of moRNAs are unknown at present ......................................... 17

3.5. Regulatory network inference ................................................................................ 18

4. Material and Methods ................................................................................................ 23

4.1. microRNAs and genes: sequences and genomic positions ..................................... 23

4.2. Biological Datasets ................................................................................................ 23

4.2.1. Multiple Myeloma dataset ............................................................................... 23

4.2.2. Acute Lymphoblastic Leukemia dataset .......................................................... 24

4.2.3. Normal and Alzheimer's parietal lobe cortex .................................................. 24

4.2.4. Normal prostate and prostate cancer ............................................................... 25

4.2.5. Multiple cancers and normal tissues dataset .................................................... 25

4.2.6. Colorectal cancer dataset ................................................................................. 25

4.2.7. Adult T-cell leukemia lymphoma dataset ........................................................ 26

II

4.2.8. SET2 cells line dataset .................................................................................... 27

4.3. Cluster analysis ...................................................................................................... 27

4.4. miRNA and gene differential expression analysis ................................................. 28

4.5. MicroRNA target predictions ................................................................................. 28

4.5.1. miRanda .......................................................................................................... 28

4.5.2. Pita .................................................................................................................. 28

4.5.3. TargetScan ....................................................................................................... 29

4.6. Regulatory network reconstruction ........................................................................ 29

4.6.1. Transcriptional regulatory network ................................................................. 29

4.6.2. Integrative analysis .......................................................................................... 29

4.6.3. Post-transcriptional regulatory networks ......................................................... 30

4.6.4. Network critical components analysis ............................................................. 31

4.7. MAGIA web-based tool ......................................................................................... 35

4.7.1. Input files ........................................................................................................ 36

4.7.2. Target predictions ............................................................................................ 37

4.7.3. Computation of interaction measure ................................................................ 38

4.7.4. Output and links to other database resources .................................................. 40

4.7.5. Bi-partite networks visualizations ................................................................... 40

4.8. Analysis of sister miRNA pairs expression ratio. .................................................. 41

4.9. Statistical analysis .................................................................................................. 41

4.10. New miRNA target prediction ............................................................................. 41

5. Results ....................................................................................................................... 45

5.1. Identification of microRNA expression patterns and definition of a

microRNA/mRNA regulatory network in distinct molecular groups of multiple

myeloma ....................................................................................................................... 45

5.1.1. Global miRNA expression profiling in MM patients ...................................... 45

5.1.2. Integrative analysis of miRNA/mRNA expression and reconstruction of a

regulatory network in MM ........................................................................................ 49

5.1.3. Critical analysis of transcriptional and post-transcriptional regulatory networks

.................................................................................................................................. 51

III

5.2. miRNAs modulation in colon cancer and metastasis development and its impact on

regulatory networks and pathways ................................................................................ 56

5.2.1. miRNA and genes expression in normal colon, colon carcinoma and liver

metastasis samples .................................................................................................... 56

5.2.2. Informativity of miRNAs and genes for samples classification and variability

in the N-T-M transitions ............................................................................................ 58

5.2.3. Differentially expressed miRNAs ................................................................... 60

5.2.4. RT-PCR miRNA expression validation ........................................................... 62

5.2.5. miRNA and genes expression profiles integration allows the identification of

most probable miRNA targets ................................................................................... 63

5.2.6. Regulatory networks modulated in tumor and metastasis development .......... 63

5.2.7. miR-182 control ENTPD5 ............................................................................... 66

5.2.8 miRNAs modulated KEGG pathways .............................................................. 67

5.3. microRNA expression in HTLV-1 infection and adult T-cell leukemia/lymphoma

...................................................................................................................................... 68

5.3.1. microRNAs with altered expression in ATLL cells ........................................ 68

5.3.2. microRNA target prediction ............................................................................ 70

5.4. MAGIA, a web-based tool for miRNA and Genes Integrated Analysis ................. 73

5.4.1. MAGIA application and testing to a case study .............................................. 73

5.5. Impact of host genes and strand selection on miRNA and miRNA* expression .... 76

5.5.1. Microarray-based expression datasets analyses description ............................ 76

5.5.2. Expression of sister mature miRNA pairs belonging to the same hairpin ....... 77

5.5.3. miRNA are hosted by long genes .................................................................... 83

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