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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

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are needed to see this picture.

The first described miRNA (2000)

lin-4 =Evolutionarily conserved

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The first described miRNA (2000)

lin-4= 22 nucleotides miRNA

Alex EcclestonQuickTime™ and a

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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)