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