Search for novel non-coding RNAs in prostate carcinoma cells

Christine Schulz

AG RNomics

Non-coding RNAs (ncRNAs)

• „classical“ ncRNAs: tRNA rRNA

• regulatory ncRNAs: miRNA (microRNA) siRNA (small interfering RNA) snoRNA (short nuclear RNA) snRNA (small nucleolar RNA) piRNA (piwi-associated RNA)

long ncRNA …

• roles in regulation of gene expression

Prostate Cancer

• 2nd leading cause of cancer death in men

Very poor prognosis for metastatic androgen-independent form

No accurate diagnostic markers to distinguish different stages

• Prostatectomy• Chemotherapy • Androgen deprivation therapy

androgen independent

Progression

androgen dependent

• Chemotherapy

Identification of novel differentially expressed ncRNAs in prostate cancer

Diagnostic markers

(differentiation between androgen-dependent and independent form)

Drug targets

Identification of novel ncRNAs using Tiling Arrays

Exonic region

Intronic region

Intergenic region

Oligonucleotide coverage (25-mer probes)

• 25-mer oligonucleotide probes cover exonic, intronic and intergenic regions of chromosomes 21 and 22 (repeats are removed)

Architecture of Human GeneChip® Tiling Arrays (chr 21 and 22)

Protein-coding gene

Cellular models for prostate cancer

LNCaP PC3 DU-145 RWPE-1

Androgen responsiveness

yes no no yes

Tiling array

Androgen-dependent

prostate cancer

Androgen-independent prostate

cancer

Epithelial prostate tissue

Experimental Approach

Cell culture, RNA isolation

ds cDNA synthesis

Fragmentation and labeling

Hybridisation

Scan

1. Tiling Array (LNCaP vs PC3)

2. Bioinformatical evaluation

• differential expression

• BLAST (RefSeqmRNA, Noncode, RNAdb) and ORF search

3. Verification of tiling array signals by RT-PCR

• Random primed and strand-specific reverse transcription PCR (RT-PCR)

Shown examples:

Results – overview

Regions for experimental verification by RT-PCR 10

Intronic 3

Intergenic 5

Overlapping intron-exon

boundaries 2

Differentially expressed regions (tiling array evaluation) 18

2

Primer design not possible (repetetive sequences, no unique sequences)

LNCaP

PC3

DU-145

RWPE-1

Antise

nse

Sense

- Prim

er

- RT

β-ac

tin

430 nt

LNCaP

PC3DU-1

45

RWPE-1

β-actin

Random primed Strand-specific

Intronic transcripts: Candidate 1 (chr 21)

RNAzEvofold

PC3

LNCaP

RefSeq (+)RefSeq (-)

mRNA-BLAST: mRNAs (95%: 1613-1921 and 4573-4887)

ncRNA-BLAST: human scAlu RNA (90%: 1619-1732 and 80%: 4768-4875)

ORF: 237 nt6701 nt

Cell adhesion molecule

288 nt

LNCaP

PC3DU-1

45

RWPE-1

β-actin

LNCaP

PC3

DU-145

RWPE-1

Antise

nse

Sense

- Prim

er

- RT

β-ac

tin

Random primed Strand-specific

Intronic transcripts: Candidate 2 (chr 21)

RNAzEvofold

PC3

LNCaP

RefSeq (+)RefSeq (-)

ORF: 150 nt

550 nt

esterase

Summary/Future Prospects

Transcripts for experimental verification by RT-PCR 10

Differential expression at expected

genomic locus2

(intronic)

Identity with transcripts

of other genomic loci

or mRNAs4

Further investigation necessary

3

Not detectable

1

• RACE-PCR

• Detection in other tissues

Acknowledgements

Group Leaders

Dr. Antje K. Kretzschmar

Dr. Jörg Hackermüller

Senior Scientific Advisors

Prof. Dr. Friedemann Horn

Prof. Dr. Peter Stadler

Group Members

Sebastian Schulz

Kerstin Ullmann

Stephan Schreiber

Katharina Schutt

Richard Schlegel

Dr. Kristin Reiche