Download microRNA: microRNA

microRNAs
Small molecules, big functions
Ali Bazargan
Brief history
•The first described microRNA, lin-4 was cloned and characterised as a
translational repressor of developmental timing from Caenorhabditis. elegans by
Lee et al (1993) and Wightman et al (1993).
•The transcript of this gene was highly unusual as it was non-coding, and
produced extremely small transcripts (22nt) from hairpin structured RNA
precursors.
•Second microRNA, let-7 was also cloned from C. elegans (Reinhart et al, 2000).
•There are currently 474 human cloned and characterised microRNA sequences
deposited in the miRBase database (http://microrna.sanger.ac.uk/sequences/)
•MicroRNAs primarily function as translational repressors by binding to
complementary target sequences in the 3’ UTR (untranslated region) of mRNA.
Brief history
•Between 10–30% of all human genes are a target for
microRNA regulation (John et al, 2004; Lewis et al, 2005).
•A single target gene often contains putative binding sites for
multiple microRNAs that can bind cooperatively ,allowing
microRNAs to form complex regulatory control networks.
•microRNAs play key regulatory roles in control of
haematopoiesis, developmental timing, cell differentiation,
apoptosis, cell proliferation and organ development as well as
in cancer, infectious disease, genetic disorders (Lin et al, 2006)
and even heart disease (van Rooij et al, 2006).
The majority of human microRNAs are encoded within introns of
coding or non-coding mRNAs whilst others are located within the
exons of non-coding mRNAs or within the 3’UTR sequence of
mRNA (Rodriguez et al, 2004).
microRNA biosynthesis and
function
•MicroRNAs are transcribed in a RNA Polymerase II-dependent manner as
large polyadenylated pri-microRNAs.
•RNAPII catalyzes the transcription of DNA to synthesize precursors of
mRNA and most snRNA and microRNA
Yang CGFR 16:397, 2005
BJH 137, 503-512 2007
Pri-microRNAs are cleaved within the nucleus by Drosha, an
RNaseIII-type nuclease, to form pre-microRNA 60–70 nucleotide
hairpin structures .
BJH 137, 503-512 2007
• Drosha requires the cofactor DiGeorge syndrome critical
region 8 gene (DGCR8) in humans (Yeom et al, 2006).
BJH 137, 503-512 2007
The pre-microRNAs are exported from the nucleus to the
cytoplasm by Exportin5 (Zeng, 2006).
•The cytoplasmic pre-microRNA is further cleaved to form an asymmetric
duplex intermediate (microRNA: microRNA*) by Dicer, another RNaseIIItype enzyme. Similar to Drosha, cofactors such as TRBP and PACT (in
humans) are necessary for Dicer activity (Lee et al, 2006).
microRNA:microRNA* duplex is in turn loaded into the miRNAinduced silencing complex miRISC
•The consequence of miRISC-loaded microRNAs is largely dependent upon
the degree of complimentarity between the microRNA and its target gene.
•It leads to either degradation of mRNA or blockage of translation without
degradation.
The choice of posttranscriptional mechanisms is not determined by
whether the small silencing RNA originated an siRNA or a miRNA but
instead is determined by the identity of the target.
Cell, Vol. 116, 281–297, January 23, 2004
Aberrant expression of microRNA
•The majority of human microRNAs are located at cancer-associated
genomic regions (Calin et al, 2004a).
•microRNA expression profiling can distinguish cancers according to
diagnosis and developmental stage of the tumour to a greater degree of
accuracy than traditional gene expression analysis (Lu et al, 2005).
•MicroRNAs play a direct role in oncogenesis as they can function as
both oncogenes (e.g. MIRN155 and members of MIRN17–92 cluster)
and tumour suppressor molecules [e.g. MIRN15A (miR-15a) and
MIRN16-1 (miR-16-1)].
•Aberrant expression of specific microRNAs has now been associated
with many types of cancer including solid and haematopoietic tumours.
BJH 137, 503-512 2007
microRNA expression in leukaemia
•Expression levels of MIRN15A and MIRN16-1, encoded within the 13q14
region, were downregulated in 75% of CLL cases that harboured this
chromosomal abnormality.
•These microRNAs were subsequently shown to target BCL2 and to induce
apoptosis in vitro, suggesting they have tumour-suppressor role in CLL
(Cimmino et al, 2005).
• MIRN16-1 negatively regulates cellular growth and cell cycle
progression (Linsley et al, 2007).
•A follow-up study (Calin et al, 2005) of 94 CLL cases, defined a
prognostically significant 13-gene microRNA signature by expression
profiling.
•Moreover two of the CLL patients were found to have germline mutations
in the MIRN16-1/MIRN15A precursor sequence that resulted in reduced
expression levels of these microRNAs both in vitro and in vivo.
TRENDS in genetics vol22, no3 March 2006
Design
• n=94 CLL pt. samples for initial dataset
• Known clinical outcome data and ZAP-70
and IgVh mutation status (retrospective)
– Zap-70 - >20% or < 20%
– IgVh status – mutated or unmutated based on
sequencing (>98% homology cutoff)
• microRNA microarray analysis of 245
miRNAs (a subset of known miRNA)
NEJM 353:1793, 2005
NEJM 353;17 0CT 2005
94 CLL
patients
Group 1
N=36
ZAP-70 +
Unmutated IgVh
Group 2
N=10
ZAP-70 +
Mutated IgVh
Group 3
N=1
ZAP-70 Unmutated IgVh
miRNA micro-array
(supervised)
13 miRNA signature (all mature):
discriminates group 1 from group 4 (p < 0.01)
Group 4
N=47
ZAP-70 Mutated IgVh
Of 13 microRNAs, 9 were significantly overexpressed in group 1,
the group with a poor prognosis
NEJM 353;17 0CT 2005
Of 13 microRNAs, 9 were significantly overexpressed in group 1, the
group with a poor prognosis
NEJM 353;17 0CT 2005
Validation
50 CLL
patients
miRNA microarray:
Applied 13 miRNA signature
algorithm
Group 1
N=36
ZAP-70 +
Unmutated IgVh
Group 4
N=47
ZAP-70 Mutated IgVh
50/50 correctly classified
94 CLL Patients
(41 treated)
Time to Initial Treatment Data
Supervised PAM
“Survival” Analysis:
Time to Initial Treatment
Short
44 +/- 39 months
Long
88 +/- 42 months
9 miRNA signature:
discriminates pts with long vs. short interval
from Dx to treatment
(ended up being a subset of 1st 13 miRNA signature)
The significance of the differences in the Kaplan–Meier curves increased if we
restricted the analyses to the 83 patients in the two main
groups (groups 1 and 4) (P values decreased from <0.01 to <0.005).
NEJM 353;17 0CT 2005
NEJM 353;17 0CT 2005
miRNA signatures identify prognostic
groups, what about pathogenesis?
Question:
Are sequence alterations in genomic DNA
responsible for observed differences in
miRNA expression?
Genomic Alterations in miRNA
• Sequenced 42 miRNA genes (including all from
•
•
identified signatures)
Germline or somatic mutations in 11/75 CLL
samples (15%)
All mutations located in 5/42 miRNAs (12%)
– miR-16-1, miR-27b, miR-29b-2, miR-187, miR-206
• 0/160 normal donors (without cancer) had these
•
miRNA genomic changes
Of 11 pts with abnormal microRNA sequence, 8
(73%) had a known personal or 1st degree
relative with cancer
NEJM 353;17 0CT 2005
microchip analysis and Northern blotting showed that CLL cells from both
patients had a substantial reduction in the expression of miR-16-1 as compared
with that of normal CD5+ cells
In Vitro Confirmation that mutations in genomic
DNA encoding miRNA affects miRNA expression
• Cloned genomic sequence encoding both miR16-1 and miR-15a (both wt and mutant C->T
+7)
• Ligated into expression vector pSR-GFP-Neo
• Transfected (lipofectamine) into 293 cells
• Assessed miRNA expression by northern
NEJM 353;17 0CT 2005
To identify a possible molecular effect of the C->T substitution, vectors
containing either the wild-type allele of the miR-15a–miR-16-1 cluster or the
mutated allele were prepared. The 293 cells, were transfected with the vectors.
NEJM 353;17 0CT 2005
•Significant association between the expression of certain microRNAs and
the expression of ZAP-70, the mutational status of IgVH, and the time
between diagnosis and initial treatment.
•A unique 13-gene molecular signature is associated with each prognostic
factor.
•microRNA expression can be included in the markers with prognostic
significance in CLL.
•microRNA signature may be relevant to the pathogenesis of CLL.
Mechanism active in CLL pathogenesis?
miR-15a and miR-16-1 induce apoptosis by
targeting bcl2
• Bcl2 protein expression is inversely correlated with miR-15a
and miR-16-1 expression in CLL samples
• Bcl2 mRNA is a direct target of miR-15a and miR-16-1
• miR-15a or miR-16-1 induce apoptosis in bcl2+ CLLcell line,
while mutant miR-16-1 c->T+7 does not
Cimmino PNAS 102:13944, 2005
Gene therapy and RNA interference
•Gene therapy, intends to provide therapeutic merit by introducing
genetic material (DNA or RNA) encoding a protein that is missing
or defective into a patient’s cells or tissues.
•A hallmark of gene therapy is the efficient delivery of these
nucleic acids via the use of shuttle vectors
•Either nonviral, such as liposomes or nanoparticles, or derived
from genetically modified viruses. ( adeno-associated virus (AAV),
adenovirus (Ad) and lentivirus)
RNAi gene therapy application
•Viral infections:
- HIV
- Hep B
- Hep C
- RSV
• Cancer
• Neurodegenerative disorders:
- Spinocerebellar Ataxias
- Huntington disease
- alzheimer disease
• Ocular disorders (Macular degeneration)
• Stem cell biology and therapy
ASH education book 2007
Summary
• miRNA may act as tumor suppressors or oncogenes
• miRNA profiles distinguish human tumor types
• miRNA signature correlates with ZAP-70/Ig Vh
•
•
•
mutation status prognosis and length of time to
initial treatment in CLL
Alterations in genomic DNA encoding miRNA exist in
CLL pts, and in 16-1 causes decreased miRNA
miR-15a and miR-16-1 directly target bcl2, and their
absence may contribute to the pathogenesis of CLL
miRNA or miRNA-like sequences may be used
therapeutically to target bcl2 or other oncogenes in
the future
Cimmino PNAS 102:13944, 2005
Indications for Treatment based on
Current Knowledge – IWCLL Guidelines
(1996)
• Advanced stage
– Improved survival with therapy restricted to Rai III/IV
stage or rapid progression of disease
• Symptoms
– Decreased PS
– Debilitating constitutional Sx
– Complications/Sx from spleen, liver, LN enlargement
• Disease activity
– Lymphocyte doubling time < 6 months
– Cytopenias (BM involvement or AI)
– Rapid LN enlargement
Binet et al. Blood DOI 10.1182, 2005