-
MicroRNAs and Cancer
MicroRNAs are an abundant class of small (20-25 nucleotides)
non-protein coding RNAs that function as negative gene
regulators.
The human genome contains up to 1000 microRNAs which constitute
approximately 1-5% of the expressed genes.
Over half of microRNA genes (52.5%) are located in or near
fragile sites or cancer-associated genomic regions
-
The discovery of microRNAs The founding member of the miRNA
family, lin-4, was identified in C. elegans through a genetic
screen for defects in the temporal control of post-embryonic
development (loss of function mutations).
1993
-
Gene structure and microRNA gene transcription
-
The biogenesis of microRNAs The linear canonical pathway of
microRNA processing
Nature 2010
-
The microRNA biogenesis factors
-
Dicer is essential for mouse development
brachyury Oct 4
morphology
Embryo 7.5
-
RISC formation and function
Components: -Argonaute (AGO)-family proteins (AGO1-4) -Gemin 3-4
Helicases
-
Domain organization of Argonaute and GW182 proteins
-
Mechanisms of post-transcriptional regulation by microRNAs
-
Principles of microRNA-mRNA interactions
perfect and contiguous base pairing of miRNA nucleotides 2 to 8,
representing the seed region (shown in dark red and green bulges or
mismatches must be present in the central region of the miRNAmRNA
duplex, precluding the Argonaute (AGO)-mediated endonucleolytic
cleavage of mRNA. there must be reasonable complementarity to the
miRNA 3 half to stabilize the interaction
-
Possible mechanisms of the microRNA-mediated
post-transcriptional gene repression in animal cells
-
P-bodies and stress granules
Decapping enzyme complex DCP1-DCP2 Decapping activators RCK/p54,
RAP55, EDC3 Deadenylase complex CAF1-CCR4-NOT 5-3 exonuclease XRN1
Other proteins involved in nonsense-mediated mRNA decay and other
mRNA degradation pathways.
-
Possible interplay between RNA binding proteins and
micro-ribonucleoproteins interacting with the mRNAs 3 UTR
-
MicroRNA editing
Editing is defined as a post-trascriptional change of RNA
sequences by deamination of adenosine (A) to inosine (I), altering
the base-pairing and structural properties of the transcript.
Editing of miRNA transcripts by ADARs (adenosine deaminases
acting on RNA) was first described for miR-22 followed by miR-151,
miR-197, miR-223, miR-376a.. Consequences. In primiR-142, A-to-I
editing inhibits its cleavage by endonuclease Drosha and results in
its degradation by ribonuclease Tudor-SN, which preferentially
cleaves double-stranded RNA containing inosine-uracil pairs.
In prmiR-151 editing abolishes its cleavage by Dicer in the
cytoplasm.
In primiR-376 a single A-to-I change redirects the mature miRNA
to a new target, resulting in altered protein expression in
mice.
To be established. Predominantly nuclear or cytoplasmic events?
Do they occur on the primiR or in the premiR?
-
Regulation of pri-miRNA processing
The microprocessor complex Drosha-DGCR8 cleaves the pri-miRNA
releasing the pre-miRNA
Some miRNAs require additional specificity factors (for example
RNA helicases p68 and p72) for efficient cleavage
-
Regulation of pri-miRNA processing
Interaction of pri-miR-18a with heterogeneous nuclear
ribonucleoprotein A1 (hnRNP A1) facilitates cleavage of this
specific miRNA by Drosha
TGF-beta signalling induces SMAD binding to the miR-21 precursor
and enhances its efficient processing by Drosha
-
Mirtrons: splicing replaces Drosha cleavage
Splicing can replace Drosha processing if the released and
debranched intron (mirtron) has the length and haitpin structure of
a pre-miRNA
-
Lin-28 regulates let-7 processing and precursor stability
Lin-28 is a stem-cell-specific regulator of let-7 processing
that uses multiple mechanisms
-
Regulation of microRNA processing factors
a. DGCR8 enhances the protein stability of Drosha b. Drosha
cleaves two hairpin structures in the DGCR8 mRNA, which
is subsequently degradated
-
microRNA maturation in the cytoplasm
AGO2 mediates pre-miRNA cleavage generating the ac-pre-miRNA
-
a. Serine 387 phosphorilation of Ago2 by p38 under cellular
stress conditions regulates its localization to P-bodies
b. Hydroxilation of Pro 700 by the type I collagen
prolyl-4-hydroxylase affects the stability of human Ago2
Argonaute proteins: regulators and effectors
a b
-
Pumilio-mediated regulation of p27 silencing by
miR-221/miR-222
-
MicroRNA identification Experimental approaches:
Cloning and sequecing endogenous small RNAs of 21-25 bp long (on
the basis of characteristics of Dicer cleavage, temporally and
spatially regulated expression and, in many cases, evolutionary
conservation)
Bioinformatic predictions (on the basis of pre-miRNA hairpin
structures and sequence conservation throughout evolution i.e.
miRScan and miRSeeker )
microRNA Registry (more than 500 in human genome)
(http://microRNA.sanger.ac.uk)
-
Functional characterization of microRNAs
Approches:
Forward Genetic Expression Studies (Reverse Genetic)
Bioinformatic predictions: TargetScan (Lewis et al.) miRanda (john
et al.) DIANA-MicroT (Kiriakidou et al.) PicTar (Krek et al.)
Algorithm for predicting vertebrate miRNA targets on the basis
of different criteria Experimental validation of potential targets
(luciferase assay)
-
miR-15 and miR-16 in Chronic Lymphocytic Leukemia
Locus 13q14 (30 kb)
-
miR-15 and miR-16 induce apoptosis by targeting BCL2
-
High-throughput tecniques for miRNA profiling
Solid-phase array-based platform Semiquantitative Requires
transcript amplification/labeling Cross-hybridization among miRNAs
of the same family
Flow-based/Liquid-phase array
Increse specificity Higher sensitivity Technically demanding
Validation by a second tecnique, such as Northern blot or
quantitative Realt Time PCR
-
An oligonucleotide microchip for genome-wide microRNA profiling
in human and mouse tissues
-
MicroRNA expression profiles classify human cancers
-
Cause of abnormal MicroRNA expression
1. Chromosomal abnormality
2. Epigenetic changes
3. Mutations and SNPs
4. Defects in the miRNA biogenesis machinery
-
1. MicroRNAs exhibit high frequency genomic alterations in human
cancer
CGH frequency plots of 227 ovarian cancer (stars indicate miRNA
genes)
aCGH data of all genomic loci containing miRNAs in ovarian
cancer, breast cancer, and melanoma specimens
-
Lung cancer patients carrying the hsa-mir-196a2 rs11614913 CC
genotype had a lower survival than the patients carrying TT/CT
genotypes, especially among stage I/II patients.
3. Functional role of SNPs in miRNA sequence: the case of
non-small cell lung cancer
-
4. Post-trascriptional regulation of microRNAs
miRNA expression during mouse development. Red bars represent
mature miRNA, and blue bars represent primary transcript.
miRNA expression in primary tumors. There is no correlation
between primiRNA mature expression in the tumor samples.
-
4. Reduced expression of Dicer associated with poor
prognosis
in lung cancer patients
-
E2F1 Regulates miR-106b-25 Expression
E2F1 Is a Target of miR-106b and miR-93
miR-106b and miR-93 Repress p21
Overexpression of miR-106b and miR-93 Interfere with
TGFb-Dependent G1/S Cell-Cycle Arrest
4.Regulation of miRNAs by transcription factors
E2F1 is a master regulator of cell cycle that promotes the G1/S
transition transactivating a variety of genes involved in
chromosomal DNA replication, including its own promoter TGFb is a
cytokine playing a major role within the so-called morphogenetic
program, a complex system of crosstalk between the epithelial and
the stromal compartments that guides gastrointestinal cells toward
proliferation, differentiation, or apoptosis
-
MicroRNAs can function as tumor suppressors and oncogenes
-
Reduced accumulation of miR-143 and miR-145 in Colorectal
Neoplasia
-
MicroRNA-21 is an antiapoptotic factor in human glioblastoma
-
Suppression of miR-21 results in caspase activation and
increased apoptosis
-
Let-7 influences Ras expression in human cells
-
The 3UTR of Nras and Kras enable let-7 regulation
-
A microRNA polycistron as a potential human oncogene
miR-17-92 cistron is located at 13q31, a genomic locus that is
amplified in cases of diffuse large B-cell lymphoma, mantle cell
lymphoma, primary cutaneous B-cell lymphoma and several other tumor
types.
-
Overexpression of the miR-17-19b cluster accelerates
c-myc-induced lymphomagenesis in mice
-
c-Myc-regulated microRNAs modulate E2F1 expression
-
miR-17-5p and miR-20a regulate E2F1 translational yield
-
Molecular mechanism of microRNA-involved cancer pathogenesis