Transcript What is a miRNA? - Science Mission

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Günther Weber
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
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
Part 1
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)
The first described miRNA (2000)
Lin(abnormal
cell LINeage)-4
L1
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L2
L3
L4
The first described miRNA (2000)
lin-4 =Evolutionarily conserved
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The first described miRNA (2000)
lin-4= 22 nucleotides miRNA
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represses accumulation of
LIN-14 protein
Alex Eccleston
Biogenesis
•
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?
•
•
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-Transcriptional
Gene 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 regulation
Contrary 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
miRNA registry
Oct 2008:
770 miRNA
H sapiens,
+ mouse, rat
Expression Patterns
Identification Methods
• Microarray – tissue variability
• Northern blot, RT-PCR, others
• Bioinformatic predictions
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
Transfection
with
miR-124
Transfection
with
miR-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 used
to 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.
Classification of tumours
of unknown origin
• 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.
Observation both on healthy tissue and cancer:
The pattern of expression of miRNA is
more characteristic for a tissue and its development
than the expression of mRNA.
1 miRNA can control 100 mRNA
To be examined: how important is miRNA expression
to differentiation and tissue development?
for cancer: how much can a miRNA contribute
to cancer development and progression?
Are miR rarely or often affected in cancer development?
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
Can miRNA work like an oncogene?
Can miRNA be tumor suppressors?
first circumstancial evidence
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 MEG01 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 can
affect the expression of multiple other genes.
The expression is characteristic for specific
tissues and cancer forms.
Some miRNAs are located at fragile chromosome
sites - chromosome breakages can thus easily alter
their expression levels.
Part 2
Can we use small RNAs against cancer?
What is the potential for RNAi
in cancer therapy?
Summary of the RNAi mechanism
Proof of concept:
Injection of 50µg siRNA into
tailvein of EGFP-transgenic
mice
downregulates luciferase
in various organs,
however not
in all cells in the organ
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 system
you 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.
alternative delivery: packaging in particles. Coating possible
to targeting the particles to cancer cells
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
Figure 5. Effect of long-term delivery of siRNA formulations on growth of metastasized
EFT in mice
Hu-Lieskovan, S. et al. Cancer Res 2005;65:8984-8992
Copyright ©2005 American Association for Cancer Research
Premature summary:
Gene therapy in general has not too much advanced
in the past years. There are successful, but few means
how 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 general
and RNAi in gene therapy in particular
should not be underestimated.
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 suppressors
Developmental Biology, 302, 1-12 (2007) (Review)
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!)
databases:
http://microrna.sanger.ac.uk/sequences/index.shtml
http://www.ma.uni-heidelberg.de/apps/zmf/argonaute/