17b micrornas and cancer - copia

8/13/2019 17b MicroRNAs and Cancer - Copia

1/34

MicroRNAs

andCancer


2/34

!Although a large proportion of the DNA in the genome istranscribed into RNA, only a very small amount

(approximately 2%) is used to produce functional proteins.

! Until recently the remaining portion of the genome wasthought to be of less importance.

! One category of these so-called non-coding sequences ofRNA, the microRNAs (miRNAs), now appears to beimportant in the regulation of many fundamentalcellular processes.

! Deregulation of miRNA levels in the cell has also beenlinked to cancer developmentin humans.


3/34

! MicroRNAs are small non-coding RNAs of 1924nucleotides in length that downregulate gene expressionduring various crucial cell processes such as apoptosis,differentiation and development .


4/34

! The first miRNA was discovered in1993 and isolated by Ruvkun and

Ambros in a study on the nematodeCaenorhabditis Elegans.

! Subsequently, hundreds of miRNAshave been identified in a wide range ofplant and animal cells.

! Estimates based on computersimulations suggest that the totalnumber of different miRNA sequences inhumans might approach 1000.


5/34

! Like C. elegans lin-4 and let-7, most miRNAgenes come from regions of the genome quite

distant from previously annotated genes, implyingthat they derive from independent transcriptionunits

!A sizable minority (e.g., about a quarter of thehuman miRNA genes) are in the introns of pre-mRNAs. These are preferentially in the sameorientation as the predicted mRNAs, suggestingthat most of these miRNAs are not transcribedfrom their own promoters but are insteadprocessed from the introns


6/34

! other miRNA genes are clustered in the genome withan arrangement and expression pattern implying tran-

scription as a multi-cistronic primary transcript.Although the majority of worm and human miRNAgenes are isolated and not clustered, over half of theknown Drosophila miRNAs are clustered.

! nearly all of the cloned miRNAs are conserved inclosely related animals, such as human and mouse, orC. elegans and C. briggsae

! many are also conserved more broadly among theanimal lineages


7/34

The two candidate RNA polymerases for pri-miRNA transcription

are pol II and pol III.

! the pri-miRNAs can be quite long, more than 1 kb, which is longerthan typical pol III transcripts.

! these presumed pri-miRNAs often have internal runs of uridineresidues, which would be expected to prematurely terminate pol IIItranscription.

! many miRNAs are differentially expressed during development,as is observed often for pol II but not pol III products.

!fusions that place the open reading frame of a reporter proteindownstream from the 5 portion of miRNA genes lead to robust

reporter protein expression, suggesting that miRNA primary

transcripts are capped pol II trascripts.

miRNA transcription


8/34

MiRNAs are initially transcribed by RNA Polymerase II (pollII) into a longer primary transcript (pri-miRNA) of several

kilobases in length.

These RNAs are capped, polyadenylated and subsequentlycleaved in the nucleus by the enzyme Drosha to liberateanother precursor of 6075 nucleotide (pre-miRNA)

The miRNA precursor (pre-miRNA) is exported to thecytoplasm by exportin 5 and it is further processed by theenzyme Dicer, resulting in a mature product of 19 24

nucleotide duplex, one strand of which is incorporated intoan effector complex called RNA-induced silencing complex(RISC)

miRNA biogenesis


9/34

The strand that is incorporated into the RISC is the maturemiRNA, whereas the opposite strand is eliminated bycleavage or a bypass mechanism.

MiRNAs regulate their targets by direct cleavage of themRNA or by inhibition of protein synthesis, according to the

degree of complementarities with their targets.

miRNA biogenesis


10/34

miRNA biogenesis


11/34


12/34

!A connection between miRNAs and cancer was suggested not longafter the initial discovery of mammalian miRNAs.

! First, Calin et al. found that the miRNAs miR-15and miR-16werelocated in an area of chromosome 13 (13q14) that is frequentlydeleted or missing in B cell chronic lymphocytic leukaemia (CLL) cells.

! It was shown that miRNA genes were frequently located atfragile sites, regions of loss of heterozygosity, regions ofamplifications or common chromosomal breakpoint regions.

The genetic damage that results from these molecular lesions can leadto specific cancers.

! Subsequently, Michael et al. first reported altered levels ofmiRNAs in human solid tumours (colonic and rectal adenomas andcarcinomas) compared with normal tissues. Altered levels of miRNAshave also been detected in breast cancer, Burkitts lymphoma,malignant brain tumours, thyroid cancer, lung cancer and

hepatocellular carcinomas


13/34


14/34

! Microarray experiments have revealed both increasesand decreasesin the production of various miRNAs in cancer. The expression patternseems to be tissue specific: different types of tumour have distinctivepatterns of miRNA expression

! If the levels of miRNAs in cancer cells, relative to normal tissues, aredifferent, it is important to identify those genes that are controlled bythese miRNAs and to see how these altered concentrations influence thedevelopment of a malignancy.

! Efforts have been made to predict and confirm possible human genesthat are controlled (targeted) by these miRNAs.

! Computer-based prediction models have been developed, based onknown complementary nucleotide sequences in miRNAs and thepredicted mRNA sequence of specific target genes.

! Several relevant target genes, which either promote (oncogenes) orsuppress (tumour suppressor genes) tumour development, havebeen identified.


15/34

Acts on/as

Tumor suppressor genes

Acts on/as

Oncogenes

miRNA


16/34


17/34

Loss of tumor suppressor function


18/34

The miRNAs, miR-15a and miR-16-1, can be considered to havetumour suppressing activity. Low levels of these miRNAs were associatedwith high Bcl-2 (anti-apoptotic B cell lymphoma2 protein) levels in CLLcells and ongoing cell growth, whereas high levels were associated withapoptosis

Another miRNA with tumour suppressing properties is let-7. Reducedlevels of let-7 were frequently seen in human lung cancers, in vitro and invivo, compared with levels in corresponding normal lung tissues. Let-7controls the activity of the critical human oncogene RAS. Lung tumour

tissues with reduced levels of let-7 had significantly increased levels ofRAS protein. The mRNA sequence of the RAS oncogene had let-7complementary binding sites, enabling let-7 to attach to RAS mRNA andprevent its translation into protein. Alternatively a low level of let-7 allowsthe RAS gene to function in an uncontrolled manner.

Loss of tumor suppressor function


19/34

Function as oncogene


20/34

Increased levels of the miRNA miR-21have been found in glioblastomascompared with levels in normal brain tissue. Reducing the level of miR-21in cultured glioblastoma cells led to increased cell death. It appears thatmiR-21 can act as a tumour promoting agent, and that overproductioncould contribute to the development of this tumour.

The miR-17 cluster of miRNAs produces increased concentrations ofmature miRNAs in lymphomas with amplification of chromosomal region13q31. Elements of this miRNA cluster are likely to include theunidentified cancer susceptibility gene(s) that are located within this 13qregion. Overproduction of miR-17-19b, in a murine lymphoma model,

accelerated the development of lymphoma.

Function as oncogene


21/34


22/34


23/34

Clinical relevance of microRNAs

There is evidence that miRNAs are involved in cancer development.Mutations in five different miRNA genes were detected in tumour

samples from 11 patients with CLL

There is some evidence of correlations between levels of miRNAexpression and clinicopathological parameters in breast and lungcancers. Changes in miRNA levels also appear to correlate withsurvival.

Reduced levels of Dicer, the cytoplasmic enzyme involved in theproduction of miRNAs, correlated with a poor grade of differentiation and

worse prognosis in lung cancer.

2. Mutations or SNPs in cancer-re lated genes, which have

previously been thought to beunimportant, may cause diseasethrough altered recognition bymiRNAs.

1.It is therefore possible thatmutations in miRNA genes

play a role in fami l ia lclustering of cancer.


24/34

Currently, almost all of the miRNA-related studies oncancers based on the different expression profile of miRNAsin cancer cells vs. normal cells. Thus, methods used for

detecting mRNA expression can also be used in studies onthe potential role(s) of miRNAs in cancers.

Principle apporaches for studying thefunction of miRNAs in cancer


25/34

Up- or down-regulated expression of miRNAs:

Up- and/or down-regulated expression of the candidatemiRNAs is a good approach to study the function ofmiRNAs in cancer pathogenesis.

Knockdown or overexpression of a specific miRNA allowsto study the specific roles of the miRNA in cancerinitiation and development

There are several methods to conduct this study, such asantisense inhibitors, transgenics, specific promoters, andpoint mutants.

Principle apporaches for studying thefunction of miRNAs in cancer


26/34

Antisense inhibitors:

In this strategy, an artificial antisense RNA competeswith cellular mRNAs to bind miRNAs.

The antisense RNA pairs with the miRNA and inhibits themiRNA function.

This has been adopted by two independent researchgroups to sequence-specifically inhibit miRNA- andsiRNA-induced RNA silencing, and inhibit four miRNAs invivo by modified antisense RNAs


27/34

Point mutations:

Point mutants of miRNAs or their targets can also

beemployed to study the function of miRNAs in cancers.

One obvious advantage of point mutants is to study the

direct interaction of miRNAs and their targeted genes.

Several studies have shown that the seed sequence is

important for miRNAs to recognize their targets, and

increasing the mismatch in the seed sequences willsignificantly decrease the gene regulation function ofmiRNAs


28/34


29/34

Northern blot analysis:

is a reliable technique to detect gene expression at themRNA level; it is widely used in gene expression analysis.

Early on, it was adopted to study the expression of miRNAgenes and now is used as a method for detecting

miRNAexpression in cancer cells.

For example, Hayashita et al. found that the miR-1792cluster is significantly overexpressed in lung cancer,especially with small-cell lung cancer, when compared

with miRNA expression in normal cells


30/34

Real time PCR:

Real-time PCR can also be employed to quantify miRNA

expression profiles and study the potential function of miRNAs

in cancer pathogenesis.

Real-time PCR recently was employed to measure miRNAprecursors and to study the expression of 23 miRNA precursorsin six cell lines.

More recently, the real-time PCR assay was expanded to 222miRNA precursor analysis in human cancer cell lines;differentexpression profiles of miRNA precursors in human

cancers do exist.

These PCR-based analyses quantify miRNA precursors and notthe active mature miRNAs. The relationship between pri-miRNAand mature miRNA expression has not been thoroughlyaddressed. This relationship is critical in order to use real-time

PCR analysis to study the function of miRNAs in cancers.


31/34

miRNA microarrays:

For cancer studies, it is important to be able to comparethe

expression pattern of all known miRNAs between cancercells and normal cells.

Thus, it is better to have methods which detect all miRNA

expression at a single time.

Two-color fluorescence-based microarray technology

(DNA microarray) has been widely used to detect gene

expression simultaneously.

Several laboratories have modified DNA microarraytechnology to form miRNA microarray technology.


32/34


33/34

Increasing knowledge of miRNA functions in human malignancy could lead tonew therapeutic strategies. It has recently been shown that a novel class

of chemically engineered oligonucleotides, termed antagomirseffectivelysilences endogenous miRNAs in vivo: Short antisense molecules, whenintroduced into a cell, bind with the available matching miRNAs, preventingthem from binding, and inhibiting, their target mRNA sequences.

An antisense Oligonucleotide, the antagomir of miR-16 (a highlyexpressed miRNAs in all tissues) could silence miR-16 in all tissues except

the brain.

Other modified oligonucleotides such as locked nucleic acid (LNA)-modified oligonucleotides can inhibit endogenous miRNAs, leading toupregulation of the target protein

Furthermore, it was revealed that miRNA expression profiles enableresearchers to successfully classify poorly characterized humantumors that can not be accurately classified by mRNA expression profiles.These results show the possibility that miRNAs have clinical benefits as not

only therapeutic targets but also a tool for cancer diagnosis.

Future strategies


34/34

The end

17b micrornas and cancer - copia

Documents