small rnas and their significance in cancer small rna is much more than rnai small rnas do more than...
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Small RNAs and their significance
in Cancer
Small RNA is much more than RNAi
Small RNAs do more than degrading one target mRNA
Small RNA is used in general for gene regulation.The molecules are similar with siRNA but may have a different regulatory effect
Günther Weber
Topics
What are small RNAs?
Where do they come from?
What do they do/why do they exist?
What is their importance in cancer?
and about RNAi:
Can we use them against cancer?
small RNA, summary:
natural production as hairpin RNA
processing with Dicer complex
to siRNA or miRNA
use either in RNA interference or for
transcriptional/translational control
What is a miRNA?
• Small single stranded RNAs (21-25 nucleotides) but derived from larger precursors (double stranded RNAs)
• Non-coding sequences
• Form imperfect stem-loop structures (hairpin)
• Hybridize by incomplete base-pairing to several sites in the 3’-untranslated regions of target mRNAs
• Negative regulators of gene expression (Postranscriptional and Translational regulation )
• Role in disease still in research ( cancer)
Part 1
The first described miRNA (2000)
Lin(abnormal
cell LINeage)-4
L1
L2
L3
L4
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
The first described miRNA (2000)
lin-4 =Evolutionarily conserved
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
The first described miRNA (2000)
lin-4= 22 nucleotides miRNA
Alex EcclestonQuickTime™ and a
TIFF (Uncompressed) decompressorare needed to see this picture.
represses accumulation of LIN-14 protein
• RNAse III enzymes
- Drosha (nucleus)
- Dicer (cytoplasm)
• Both enzymes involved in the generation of siRNA
• RISC = RNA-induced silencing complex (contains miRNPs and Argonaute family proteins)
• RISC = Degradation/Silencing?
Biogenesis
Molecular Hallmarks
~ 70 nucleotides
~ 22 nucleotides
A principle of miRNA:Only part of miRNA is complementary to its target, thus its specificity is more limited.Therefore one miRNA can have different targets
Match with target (degree of complementarity)
= Prob. of degradation
• Possibly each miRNA may target multiple genes
• miRNA = or siRNA ?– Biochemically
indistinguishable– Single vs double-stranded– Repression vs degradation?
• Post-TranscriptionalGene Silencing (PTGS)versus TransIational Inhibition
Function in Translation
mRNA-specific Regulation of Translation
Control by Micro RNAs
The “head” is regulated by the “tail”miRNA interferes in this regulationContrary to RNAi, the mRNA is not degraded
Practical distinction PTGS vs translational repression:
PTGS: mRNA AND protein levels reduced/disappeared
Transl. repression: Protein reduced, mRNA maintained
Useful methods:
ELISA/Western blot analysis plus qPCR/Northern blot
Oct 2008:770 miRNAH sapiens,+ mouse, rat
miRNA registry
Identification Methods
• Microarray – tissue variability
• Northern blot, RT-PCR, others
• Bioinformatic predictions
Expression Patterns
miRNA microarray design
• selection of miRNAs• 161 human• 84 mouse• 3 arabidopsis
• 40-mer oligos spotted on array
Experimental procedures
• 2.5 µg total RNA • Incubated with a 3'-(N)8-(A)12-biotin-(A)12-biotin-5‘
oligonucleotide primer• 1st strand synthesis• Hybridisation at 25°C o.n.• Detection using a streptavidin-Alexa647 conjugate
miRNA expression in normal human tissues
Bead-based detection
• purification of miRNA on PAGE
• adaptor ligation• reverse transcription
and amplification with PCR
• denaturation• hybridisation to beads
Correlation between
bead data and Nothern Blot
data
Hierarchial clustering of miRNA expression
Conclusions
• Different tissues have distinctive patterns of miRNome expression (defined as the full set of miRNA in a cell) with each tissue presenting a specific signature.
• Some miRNAs are highly expressed in only one or a few tissues.
mRNA targets of miRNAs
Methodology
• To investigate the influence of miRNAs on transcript levels
• miRNA transfection into human cells microarray
TransfectionwithmiR-124
TransfectionwithmiR-1
Conclusions
• miR-124 shifts towards brain expression profile• miR-1 shifts towards muscle expression profile• ~100 messages were downregulated after 12 hs• miRNAs can also reduce transcripts levels (not only
proteins)• Help to define tissue-specific gene expression in
humans
Can the expression of miRNAs be usedto characterize human cancer?
Most miRNAs have a lower expression level in tumours compared with normal tissue…
…and even lower expression levels in poorly differentiated tumours
Classification of tumours of unknown origin
• 2-4 % of all cancer diagnoses represent cancers of unknown origin or diagnostic uncertainty
• 17 poorly differentiated tumours where the histological appearance was non-diagnostic (but for which diagnosis was established by anatomical context) were used (5 breast, 1 colon, 8 lung, 3 ovary).
• Training set based on 68 samples from 11 tissue types.
• Prediction of tissue origin using a probabilistic neural network (PNN) method:– 11 out of 17 samples correctly predicted
(2 breast cancer samples and 4 lung cancer samples erroneously predicted).
– 1 out of 17 samples correctly predicted using mRNA based classification.
Classification of tumours of unknown origin
Observation both on healthy tissue and cancer:
The pattern of expression of miRNA is more characteristic for a tissue and its developmentthan the expression of mRNA.
1 miRNA can control 100 mRNA
To be examined: how important is miRNA expressionto differentiation and tissue development?
for cancer: how much can a miRNA contributeto cancer development and progression?
Correlation between Fragile chromosome sites and location of miRNA genes
Calin G. A. et.al. PNAS 2004;101:2999-3004
Copyright © 2004, The National Academy of Sciences
Are miR rarely or often affected in cancer development?
Can miRNA work like an oncogene?
first circumstancial evidence
Can miRNA be tumor suppressors?
The majority of CLL samples express lower levels of miR16 and miR15 as normal CD5+ cells. The LOH status for the presented samples is shown as: +/+, heterozygosity; +/−, LOH; −/−, homozygous deletion; NI, not informative; ND, not done. As normalization controls, we used staining with ethidium bromide of Northern gels.
Bcl2 protein expression is inversely correlated with miR-15a and miR-16-1 miRNAs expression in CLL patients
Cimmino A. et.al. PNAS 2005;102:13944-13949
Copyright © 2005, The National Academy of Sciences
BCL2 is a target of miR-15 and miR-16.(A) Transfection of miR-15a/miR-16-1 cluster in MEG-01 BCL2+ leukemia cells is followed by a significant reduction in protein levels
Cimmino A. et.al. PNAS 2005;102:13944-13949
Copyright © 2005, The National Academy of Sciences
Apoptotic evaluation determined through comparison of TUNEL-positive apoptotic nuclei and total number of cells
Cimmino A. et.al. PNAS 2005;102:13944-13949
Copyright © 2005, The National Academy of Sciences
Is the expression of certain miRNA characteristic for cancer?
Examples: miRNA influencing known cancer-related genes
MiRs as cancer players
Calin G. A. et.al. PNAS 2004;101:2999-3004
Copyright © 2004, The National Academy of Sciences
Summary:miRNA are regulating elements for gene expression,both on the levels of transcription and translation.
miRNA expression can be altered in cancer, which canaffect the expression of multiple other genes. The expression is characteristic for specifictissues and cancer forms.
Some miRNAs are located at fragile chromosome sites - chromosome breakages can thus easily altertheir expression levels.
What is the potential for RNAi in cancer therapy?
Part 2
Can we use small RNAs against cancer?
Summary of the RNAi mechanism
Injection of 50µg siRNA into tailvein of EGFP-transgenic mice
downregulates luciferase in various organs, however notin all cells in the organ
Proof of concept:
RNAi can be used to suppress unwanted alternatively spliced transcripts
RNAi can be used to suppress exon skipping
RNAi can be used to suppress transcripts with point mutations
Problems for RNAi
1. specificity sometimes “hyped”:side effects as micro RNA quite possible “The almost ideal specificity of RNAi has shown not to hold
entirely true in reality”
2. efficiency of delivery systemyou can’t inject enough siRNA for treatment
3. specificity for target cell to be questioned -you don’t want to shut off a vital gene in a healthy cell.
Fig. 2. The left hand side shows various non-targeted or targeted in vivo delivery strategies for RNA-based siRNA drugs. The right hand side shows a schematic diagram of either a DNA-based pol-III or pol-II promoted shRNA expression cassettes. These shRNA expression units can by delivered by viral or non-viral methods to the target tissue, as illustrated by gene transfer using lentiviral vector technology.
Figure 2. Growth of xenograft tumors. Each point represents the mean volume ± standard deviation on the X-axis. Point 1 represents the day of DNA injection; point 4 was the time when the mice were sacrificed. *P<0.001, VEGF-C-siRNA group vs control group.
Figure 1. Schematic illustration of the delivery system. A, components of the delivery system. The CDP condenses siRNA and protects it from nuclease degradation. The AD-PEG conjugate stabilizes the particles in physiologic fluids via inclusion compound formation. The AD-PEG-transferrin (Tf-PED-AD) conjugate confers a targeting ligand to particles, promoting their uptake by cells overexpressing the cell-surface transferrin receptor. B, assembly of the nontargeted and targeted particles. For nontargeted particles, CDP and AD-PEG are combined and added to siRNA to generate stable but nontargeted polyplexes. For targeted particles, CDP, AD-PEG, and AD-PEG-transferrin are combined and added to siRNA to generate stable, targeted particles
alternative delivery: packaging in particles. Coating possibleto targeting the particles to cancer cells
Copyright ©2005 American Association for Cancer Research
Hu-Lieskovan, S. et al. Cancer Res 2005;65:8984-8992
Figure 5. Effect of long-term delivery of siRNA formulations on growth of metastasized EFT in mice
Premature summary:
Gene therapy in general has not too much advancedin the past years. There are successful, but few meanshow to treat cancer by gene therapy.
RNAi does not belong to them. We are still in the experimental phase using RNAi.
The problems for gene therapy in generaland RNAi in gene therapy in particularshould not be underestimated.
databases:http://microrna.sanger.ac.uk/sequences/index.shtmlhttp://www.ma.uni-heidelberg.de/apps/zmf/argonaute/
Lee R, Feinbaum R, Ambros V.: A short history of a Short RNA.Cell, S116, S89-S92 (2004) (highly recommended to learn how to do real Science!)
Reviews for those who liked the story:
Barthel DP: MicroRNAs: Genomics, Biogenesis, Mechanism, and Function.Cell, 116, 281-297 (2004) (Review)
Zhang B, Pan X, Cobb GP, Anderson TA: microRNAs as oncogenes and tumor suppressorsDevelopmental Biology, 302, 1-12 (2007) (Review)