Functional characterization of the DLEU2 gene

Johan Sundström

Department of Oncology-Pathology,

Cancer Center Karolinska

Master thesis in medical science with a

major in biomedicine.

Stockholm 2006

By: Johan Sundström

Supervisor: Martin Corcoran, department of Oncology-Pathology

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Department of Oncology-Pathology,

Cancer Center Karolinska

Master thesis in medical science with a

major in biomedicine.

Stockholm 2006

Functional characterization of the DLEU2 gene

Abstract

DLEU2 is one of two genes present within a 10 kb minimal region of loss at chromosome 13q14 that is deleted in ~50% of cases of chronic lymphocytic

leukemia (CLL). Another group has defined a 30 kb minimal region of loss, and it is still unclear exactly which critical elements that are disrupted by genomic

deletion in CLL.

Two microRNAs, miR-15a and miR-16-1 were found at 13q14. These microRNAs potentially regulate the expression of target genes and may function

as tumor suppressor genes.

The 30 kb minimal region of loss encompasses these two microRNAs, and studies have showed that they were downregulated in many CLL cases. However, our 10

kb minimal region of loss is telomeric to that region.

It has recently been found that an alternatively spliced transcript of DLEU2

termed DLEU2MIR has the two microRNAs, miR-15a and miR-16-1 located in

its 3' exon. We have transfected cell lines with a construct of DLEU2MIR and show by Nothern blot analyses that this up-regulates the expression of the

microRNAs. These data show that the microRNAs can be expressed through this splice form of DLEU2, suggesting that loss of the 10 kb region including the

promoter region and first exon of DLEU2 might lead to down regulation of miR-15a and miR-16-1 expression.

3

Sammanfattning

DLEU2 är en av två gener som ligger i en 10 kb region på kromosom 13q14 som

är deleterad i ungefär hälften av alla fall av kronisk lymfatisk leukemi (KLL). En annan forskargrupp har definierat en 30 kb region som minsta deleterade region

och det är fortfarande oklart vilka kritiska element som skadats vid genomisk deletion i KLL.

Två mikroRNA, miR-15a och miR-16-1 hittades vid 13q14. Dessa mikroRNA är

potentiella genreglerare och kan möjligen fungera som tumörsuppressorgener.

30 kb regionen omfattar dessa två mikroRNA och studier har visat att de var nedreglerade i många fall av KLL. Vår 10 kb region ligger emellertid telomeriskt

till denna region.

Det upptäcktes nyligen att en alternativ spliceform av DLEU2 kallad DLEU2MIR har ett 3'-exon som innehåller dessa två mikroRNA, miR-15a och miR-16-1. Vi

har transfekterat cellinjer med ett konstrukt av DLEU2MIR och visar med Northern blot-analys att detta uppreglerar mikroRNA-uttrycket. Resultaten visar

att dessa mikroRNA kan uttryckas genom denna spliceform av DLEU2, och att deletion av 10 kb-regionen som innehåller DLEU2s promotor och första exon

kanske kan leda till nedreglering av miR-15a och miR-16-1.

TABLE OF CONTENTS Abbreviations................................................................................................................ 5

Introduction .................................................................................................................. 6

Chronic Lymphocytic Leukemia ............................................................................... 6

Deletion of 13q14 ..................................................................................................... 7

microRNAs............................................................................................................... 9

microRNAs and CLL .............................................................................................. 11

Aim ............................................................................................................................ 14

Materials and methods ................................................................................................ 15

Cell cultures............................................................................................................ 15

Plasmids.................................................................................................................. 15

siRNA oligos .......................................................................................................... 16

Transfections........................................................................................................... 16

RNA isolation ......................................................................................................... 16

microRNA Northern Blot........................................................................................ 16

RT PCR .................................................................................................................. 17

Results ........................................................................................................................ 18

Discussion .................................................................................................................. 22

Results .................................................................................................................... 22

Method ................................................................................................................... 23

Relevance ............................................................................................................... 23

Acknowledgements..................................................................................................... 27

References .................................................................................................................. 28

5

Abbreviations

AIHA Autoimmune hemolytic anemia

ARE AU-rich element

ATM Ataxia Telangiectasia Mutated

B-CLL B-cell chronic lymphocytic leukemia

CLL Chronic lymphocytic leukemia

CML Chronic myeloid leukemia

DMSO Dimethylsulfoxide

DNA Deoxyribonucleic Acid

PCR Polymerase chain reaction

RISC RNA Induced Silencing Complex

RNA Ribonucleic Acid

RNAi RNA interference

SDS Sodium Dodecyl Sulfate

siRNA Short interfering RNA

SSC Sodium chloride-sodium citrate

TBE Tris-Borate-EDTA Buffer

UTR Untranslated region

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Introduction

Chronic Lymphocytic Leukemia

B-cell chronic lymphocytic leukemia (B-CLL) is the most common form of leukemia in

the Western world and accounts for more than 30% of the leukemia cases in Europe and

North America. The disease usually affects patients over the age of 60, with the median

age at diagnosis being 70 years. It is more common among males (1) and 458 new cases

of CLL were reported to the Swedish Cancer Registry in 2004 (2).

The disease is characterized by a progressive accumulation of mature B-lymphocytes in

peripheral blood. The cells express the B-cell antigens CD19 and CD20 together with

the T-cell antigen CD5, which gives them a characteristic immunophenotype used to

diagnose the condition. They express very low levels of surface immunoglobulins. The

etiology of the disease is unknown (1).

Perhaps as a result of the accumulation of non-functional B lymphocytes many CLL

patients develop hypogammaglobulinemia, which makes them immunodeficient and

susceptible to infections. Autoimmune disorders such as autoimmune hemolytic anemia

(AIHA) are also common in CLL patients. Infection is the most frequent cause of death

in CLL patients (1).

It is often thought that CLL is a very slowly progressing disease and that the cause of

death is unrelated to the disease, but this is only true for less than 30% of the cases.

CLL is a very heterogeneous disease and shows different progression in the individual

cases. Some patients die already after 2-3 years, and even though many live for 5-10

years the terminal phase has considerable morbidity from the disease itself and from

complications of therapy. Due to its heterogeneity the median survival times vary from

19 to 150 months depending on which clinical stage the patient is classified into at the

time of diagnosis (1).

7

The disease can be treated to reduce symptoms but cannot be cured by treatments

available today. Chemotherapy with alkylating agents and corticosteroids is the most

common treatment, but monoclonal antibodies and local radiation are also used (1).

Much effort has been made to find genes that might be involved in the pathogenesis of

CLL. Previous studies of common genetic abnormalities found in other diseases have

led to discoveries of important pathogenic disease genes, for example ataxia

telangiectasia mutated (ATM) (3). Knowing the molecular background of a disease is

crucial for finding therapeutic targets and for drug development. A successful example

of this is imatinib, a small molecule inhibitor of the BCR-ABL fusion protein that is

commonly found in cases of Chronic myeloid leukaemia (CML), which significantly

improved treatment of the disease (1).

Chromosomal aberrations are often found in CLL cells. The most common abnormality

is deletion of chromosome 13 at cytogenetic band q14, which is found in more than

50% of cases, followed by deletion of 11q22-23 (in 18% of cases) and trisomy 12 (in

16% of cases). Deletion of 11q22-23 is thought to inactivate the tumor suppressor gene

ataxia telangiectasia mutation (ATM) and a small percentage of CLL cases have

mutated p53 (4) but in the majority of cases the genetic cause of the disease is still

unknown. Deletion of 13q14 probably represents early clonal aberrations in CLL, and

many studies suggest that a lost or inactivated tumor suppressor gene should be present

in this region. A number of putative genes have been found in the region, but their

involvement in the pathogenesis of CLL remains to be elucidated (1).

Deletion of 13q14

45-55 % of CLL cases have hemizygous or homozygous loss of a part of chromosome

13q14.3, (5) with loss of this locus being the most common chromosomal abnormality

found in CLL. Deletions of 13q14 are also often found in mantle cell lymphoma,

prostate cancer and multiple myeloma, further suggesting that one or more tumor

suppressor genes are present in the region and involved in the pathogenesis of human

cancers (6).

8

However, there is a lack of a clearly defined minimal region of loss and different

research groups have proposed slightly variable regions (Figure 1). A small 10 kb

region of loss was found by one group and two putative tumor suppressor genes termed

DLEU1 and DLEU2 (deleted in lymphocytic leukemia 1 and 2) were mapped to this

region at 13q14.3 (7). The first exon and the promotor region of both genes were

deleted in many cases of 13q14 loss, but no small intragenic mutations or point

mutations were found in the genes in hemizygous loss cases (7). RFP2 is another

adjacent candidate tumor suppressor gene in the region (8) that is located 50 kb

centromeric of the 10 kb region of loss. Of the genes in the commonly deleted region,

only DLEU2 and RFP2 were found to be conserved at the genomic sequence level

between human and mouse (9). Since DLEU2 is not conserved at the amino acid level it

is suggested to be a non-protein-coding RNA transcript. One of its exons has a sequence

identity in an antisense fashion to exon 1A of the RFP2 gene, and could therefore be

involved in regulation of this gene, see figure 1 (10). Two microRNAs, miR-15a and

miR-16-1 are located at 13q14 (6) and it was found that they are located in an intron of

the gene DLEU2, outside the minimal region of loss (10). The question arises if the

expression of DLEU2 affects the expression of miR-15a and miR-16-1. Research in

Grandér’s lab has concentrated on this question, and an important breakthrough was the

finding of an alternatively spliced DLEU2 transcript that encodes miR-15a and miR-16-

1 within its 3' exon, see figure 1. This DLEU2 splice form will from now on be referred

to as DLEU2MIR. DLEU2MIR may theoretically function as a tumor suppressor gene

by expressing the microRNAs that downregulate oncogenic target genes.

Since the first exon and promoter region of DLEU2 is deleted in all CLL cases with

13q14 loss, this may lead to decreased expression of miR-15a and miR-16-1. Lower

levels of the microRNAs will lead to up-regulation of their target genes. The

microRNAs will be discussed in the following sections.

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Figure 1. The different minimal regions of loss as defined by Calin et al and Liu et al and the splicing of

DLEU2MIR, encoding the two microRNAs within its 3' exon and the splice form DLEU2ANTI that has a

sequence identity in an antisense fashion to exon 1A of the RFP2 gene.

microRNAs

Non-protein-coding small RNA molecules of about 22 nucleotides in length called

microRNAs (miRNAs) can down regulate gene expression by binding to target mRNA

molecules (Figure 2). The first miRNA was discovered in C. elegans in 1993 (11), and

now hundreds of miRNAs are confirmed to be encoded in the human genome and

computational studies estimate that there might be as many as one thousand miRNAs,

potentially regulating a large number of other genes (13). Most human miRNAs are

located within introns of protein-coding or non-protein-coding mRNA transcripts. There

are several studies showing that miRNA expression is correlated with various cancers,

and miRNAs are thought to function both as tumor suppressors and oncogenes. It has

also been shown that about 50% of the known human miRNAs are located in so called

fragile sites, regions that are sensitive to breaks and often are deleted or rearranged in

cancer (12).

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The miRNAs are transcribed by RNA polymerase II to produce primary-miRNAs (pri-

miRNAs) that often are several kilobases long and are polyadenylated and capped

similar to protein encoding mRNAs (13). A protein complex called Drosha then

processes the pri-miRNAs and a ~70 nucleotide loop called pre-miRNA is excised. The

pre-miRNAs are exported from the nucleus to the cytoplasm, where Dicer cleaves them

to a small double stranded miRNA, which is further processed to a mature single

stranded miRNA (13). If the mature miRNA bind with perfect sequence complementary

to a target mRNA it will induce the RNA interference pathway (RNAi) which leads to

degradation of the target mRNA through activation of the RNA-induced silencing

complex (RISC). However, if the miRNA sequence has imperfect complementarity to a

target mRNA it will down regulate the gene expression by repressing the translation

through activation of a miRISC complex. This is thought to be the most important

mechanism of miRNA gene regulation in mammals, and in this case the mRNA level

will not be affected so the effects must be detected at the protein level. The binding site

on the target mRNA is often located in the 3' untranslated regions (UTRs), but since the

sequence complementarity is imperfect it is difficult to predict gene targets. miRNAs

have highest homology at the 5' end and this part of the miRNA molecule seems to be

most important for binding to the miRISC complex and the target mRNA (12).

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Figure 2. The biogenesis of microRNAs from Esquela-Kerscher et al. 2006 (12).

microRNAs and CLL

As mentioned, it has been shown that two microRNAs, miR-15a and miR-16-1, are

located at 13q14 and there are several studies suggesting that they are involved in the

pathogenesis of CLL. Calin et al argue that they are located within a 30-kb region of

loss found in CLL (Figure 1) and show that both miRNAs are deleted or down regulated

in more than 60% of CLL patients (6). The results from this study was further

confirmed by a microarray study showing down regulation of miR-16-1 in 45% of the

CLL cases and of miR-15a in approximately 25% of the cases (14). Abnormal

expression of the murine miRNA genes mmu-mir-15 and mmu-mir-16 was found in a

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mouse model of CLL, further suggesting that the microRNAs are involved in

pathogenesis of this disease (15).

However, according to the 10 kb minimal region of loss defined by Grandérs group the

region containing the miRNA genes is not deleted in all cases. Instead, exon 1 and the

promotor region of the gene DLEU2 is lost, and since miR-15a and miR-16-1 are

located in an alternative 3' exon of DLEU2 (the DLEU2MIR splice form) this could be

the reason for their down regulation. In this study we transfect cells with a construct of

DLEU2MIR to see if this up-regulates the expression of miR-15a and miR-16-1, to

show that they are expressed through this splice form of DLEU2 (Figure 3). This

finding would also tie together the seemingly disparate findings and show that the miRs

can be lost either by a large deletion encompassing the miRs themselves, or by a small

deletion of exon 1 and the promotor region of DLEU2 that disrupts the primary

transcript through which they are expressed. Improved knowledge about the expression

and function of microRNAs in general is of course also of high scientific interest.

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Figure 3. Schematic representation of the DLEU2MIR transcript. It has an alternatively spliced exon 4

variant (4alt) that extends into intron 5 of DLEU2 including the microRNAs miR-15a and miR-16-1. The

4alt exon ends close to a polyadenylation signal indicating that it is an alternative 3' exon. The transcript

is processed by the Drosha complex in the nucleus forming the pre-miRs that are exported to the

cytoplasm for further processing by Dicer into the 22 bp mature microRNAs.

Little is known about the targets and mechanisms of miRNA action. One study shows

that miR-15 and miR-16 expression is inversely related to the level of Bcl2, an

antiapoptotic gene. Down regulation of miR-15 and miR-16 leads to up-regulation of

Bcl2 and presumably block of apoptosis in B-CLL cells. They also found

complementarity with the first 9 nucleotides of both miRNAs with a region of the Bcl2

mRNA (16). Bcl2 is up-regulated in about 85% of CLL cases, and high levels of this

anti-apoptotic protein may be an important contributor to the accumulation of the

malignant cells (1).

Another study shows that miR-16 is required for turnover of AU-rich element (ARE)

containing mRNAs. AREs are found in the UTRs of a variety of short-lived mRNAs

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such as cytokines. miR-16 carries the sequence UAAAUAUU, which is complementary

to the ARE sequence. miR-16 together with the ARE binding protein tristetraprolin

(TTP) appears to be essential for ARE-mediated mRNA degradation (17). DLEU2MIR

contains a consensus AU-rich regulatory element, a finding in common with several

mRNAs that function in this pathway and which is believed to function as a negative

feedback element.

A study by Mao et al suggests that DLEU2 is repressed by the transcription factor c-

Myc. They analyzed the mRNA expression of DLEU1 and DLEU2 in HL60 cells

without DMSO treatment (high levels of c-Myc) and with DMSO treatment (lower c-

Myc expression). According to this study DLEU1 and DLEU2 are both up-regulated in

DMSO treated HL60 cells due to a lowered c-Myc repression of the genes (18). To

investigate if the DLEU2MIR spliceform is affected we treated HL60 cells with DMSO

and measured the levels of miR-16.

We also performed siRNA knockdown of DLEU2 and Drosha to see if that would lower

the miR expression. It takes several days for cells to degrade microRNAs, so to be able

to see the effects of the siRNA knockdowns the transfected cells were split on the third

day and then retransfected as has been described by Lee et al (19).

Aim

- To study whether expression of DLEU2MIR in cell lines will increase the

expression of miR-15a and miR-16-1.

- To investigate if DMSO treatment of HL60 cells increase the expression of miR-

16.

- To knock down DLEU2 and Drosha with siRNA and see if that lowers the levels

of miR-16.

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Materials and methods

Cell cultures

The HEK293 and U2OS cell lines were maintained in Dulbecco's Modified Medium

containing 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 g/ml streptomycin

at 37 °C and 5% CO2. The MCF7 and HL60 cell lines was maintained in RPMI 1640

medium supplemented with HEPES, 10% FCS, 2 mM glutamine, 100 U/ml penicillin

and 100 g/ml streptomycin at 37 °C and 5% CO2.

Plasmids

The DLEU2MIR expression construct was created by ligating a 1.5 kb DLEU2MIR

specific PCR fragment amplified using the primers DLEU2MIRF

(5'-CTCTAACGAATTTGAATGAGGAGC-3') and DLEU2MIRR

(5'-CGTGAGCCACCGCACCTGGCTGTC-3') from a bone marrow RNA derived

cDNA template into the vector pCDNA3.1/V5-His-TOPO (Invitrogen) (Figure 4). This

construct was created by Martin Corcoran. A non-insert containing plasmid was used as

a negative control in all transfection experiments.

Figure 4. A schematic picture of the pcDNA1.3/V5-His-TOPO vector.

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siRNA oligos

The following siRNA oligos were used:

siRNA oligos against DLEU2 siRNA oligos against Drosha

DLEU2r 5'-AAUCAAAGAAGUGAGGCUGTT-3' DroshaS 5'-CGAGUAGGCUUCGUGACUUTT-3'

DLEU2exon1A 5'-AAAUCGCCAAAAAUUACUUTT-3' DroshaaS 5'-AAGUCACGAAGCCUACUCGTT-3'

DLEU2exon3 5'-GAGACCUCUACUUAGCUC-3'

siRNA oligos against green fluorescent protein (GFP) was used as a negative control.

Transfections

HEK293, U2OS and MCF7 cells were transfected using TransIt LT1 transfection

reagent (Mirus) according to the manufacturer’s protocol. Cells were transfected at ~

50% confluency with 2 μg plasmid DNA for each well in 6-well format (10 μg plasmid

DNA for a 10 cm plate). Cells were harvested 48 h after transfection.

For siRNA experiments HEK293 cells were transfected using TransIT TKO

transfection reagent (Mirus) according to the manufacturer’s instructions. Cells were

transfected on day 1, retransfected on day 2, split on day 3, retransfected again on day 4

and 5 and harvested on day 6.

RNA isolation

Cells were lysed in TRI Reagent (Ambion) for total RNA isolation according to the

manufacturer’s protocol. A small aliquot of the total RNA was run on a 1 % agarose gel

to check the integrity of the RNA.

microRNA Northern Blot

~ 5 μg of total RNA was loaded onto 15% denaturing urea-acrylamide gels run in 0.5%

TBE buffer. The gel was run for 2 h at 300 V. The gel was stained in running buffer

with Ethidium Bromide for 10 minutes, then de-stained in running buffer for 10 min and

photographed to check that the RNA has maintained integrity and was properly loaded

onto the gel. The RNA was transferred from the gel to BrightStar-Plus positively

charged nylon membranes (Ambion) in 0.5% TBE buffer for 1h at 200 mA using a

semi-dry transfer apparatus. Membranes were crosslinked with UV light for 1 minute.

Membranes were pre-hybridized in 5 ml ULTRAhyb-Oligo hybridization buffer

17

(Ambion) containing 100 μg ssDNA for 1h and then incubated overnight with a 32

P-

ATP-labelled probe. The probes used were specific for miR-16

(5'-CGCCAATATTTACGTGCTGCTA-3'), miR-15a

(5'-CACAAACCATTATGTGCTGCTA-3') and the 106 bp snoRNA U6

(5'-GCAGGGGCCATGCTAATCTTCTCTGTATCG-3') was used as a control. The

membrane was hybridized over night at 37 °C for the miR probes and at 42 °C for the

U6 probe. Membranes were then washed in 2X SSC solution with 0.1% SDS at room

temperature for 20 min (miR probes) and 30 min (U6 probe) followed by a wash in 1X

SSC with 0.1% SDS at 42 °C for 20 min (U6 only). After washes the hybridized

membranes were exposed to Fujifilm SuperRX x-ray film at –70 °C for 48 h (miR

probes) and at room temperature for 20 min (U6 probe).

RT PCR

The RNA was reverse transcribed with First-Strand cDNA synthesis using Superscript

II (Invitrogen, Life Technologies) and semi quantitative RT-PCR was carried out with

primers specific for GAPDH and DLEU2MIR. GAPDH expression was used as a

control to measure the total amount of RNA in each sample. Expression analysis of the

DLEU2MIR spliceform was performed using the spliceform specific primers DL2MF1

(5'-CTCAGCAATTCTTACCTTTCTTAC-3') and DL2MR1

(5'-GGGCAATACAACTAAATGCTTAAAC-3').

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Results

In principle, cloning of microRNA containing transcripts can be made as shown in

figure 5. A region of about 1 kb encompassing the microRNA of interest is amplified by

PCR and ligated into an expression vector. Cells are transfected with the vector, and an

up-regulation of the mature microRNA can be detected. To verify that this principle

works not only for DLEU2MIR, another microRNA called miR-301 was also cloned

and expressed.

Figure 5. A schematic representation of how a microRNA is cloned for expression.

Transfection of the miR-301 construct into HEK293 cells resulted in up-regulation of

the mature miR-301 and the pre-miR-301 (Figure 6).

microRNA

~ 1 kb

Ligation into vector

exon

19

Figure 6. Transfection of a miR-301 construct (+) and pcDNA control (-) into HEK293 hybridized with a

probe specific for miR-301.

A Northern blot for small RNA analysis differs from a conventional Northern blot. A

substantial part of the project was to set up this method at the lab and get it working

consistently, since it is not a standard technique. Because of the small size of the

microRNAs a very dense polyacrylamide gel must be used for separation. Since the

microRNAs often are present at low concentration the sensitivity must be very high.

Therefore all steps of the analysis had to be optimized. Critical aspects seemed to be

transfer conditions (time, buffer) and crosslinking of the membrane (time in UV light).

Hybridization solution and membranes suitable for microRNAs were used.

Transfection of the cell line HEK293 with a plasmid construct of DLEU2MIR resulted

in up-regulation of the microRNAs miR-15a and miR-16-1. Both the fully processed

mature microRNAs (22 nt) and the precursor pre-miRNAs (miR-15a 83 nt, miR-16-1 89

nt) are detected (Figure 7). The up-regulation shows a similar pattern for miR-15a and

miR-16-1.

pre-miR-301

miR-301

- +

20

Figure 7. Transfection of pDLEU2MIR (+) and pcDNA control (-) into HEK293 hybridized with probes

specific for miR-15a and miR-16.

The cell lines U2OS and MCF7 were also transfected with the same construct and

hybridized with a probe specific to miR-16. All show increased levels of the 22 bp miR-

16 and the 89 bp pre-microRNA (Figure 8).

Figure 8. Transfection of pDLEU2MIR (+) and pcDNA control (-) into the cell lines HEK293, U2OS and

MCF7.

DMSO treatment of HL60 cells seems to give an up-regulation of miR-16 after 24 h

(Figure 9).

HEK293 U2OS MCF7

U6

pre-miR-16

miR-16

- + - + - +

mature miRNA

pre-miRNA

miR-15a miR-16

- + - +

21

Figure 9. DMSO treatment of HL60 cells. Sample 1 was harvested at time point 0, sample 2 was treated

with 1% DMSO for 24h, sample 3 was treated with 1% DMSO for 48h.

The samples from the siRNA experiment (data not shown) were analyzed for miR-16

expression but the levels were too low to give any reliable results.

To check the expression of DLEU2MIR, RT PCR analysis was carried out using cDNA

templates from haematological cell lines (Figure 10). The analysis shows high

expression of DLEU2MIR in preB-ALL cell lines. Highest expression is seen in cell

lines RCHACV, 697, 38 , CEMO and RS4;11. The expression pattern suggests that the

gene is expressed at its highest levels in the early stage of lymphocytic differentiation, a

finding that fits in with DLEU2 being a target of BLIMP-1 which downregulates certain

genes in later stages of plasma cell development.

Figure 10. Expression of DLEU2MIR in cell lines analyzed by RT PCR. 1. U266, 2. MUT25, 3. NALM6,

4., HL60, 5. PER365, 6. KASUMI, 7. RCHACV, 8. 697, 9. REH, 10. 38 , 11. CEMO, 12, RS4:11, 13.

PER37, 14. TANOE, 15. SALPD3

miR-16

U6

1 2 3

22

Discussion

Results

The principle question in this study was whether ectopic expression of the DLEU2MIR

transcript would result in expression of the processed mature microRNAs miR-15a and

miR-16-1. Initial experiments showed similar expression patterns of miR-15a and miR-

16-1, and the further analyses were focused on miR-16-1. Transfection of the construct

DLEU2MIR into the cell lines HEK293, U2OS and MCF7 resulted in rapid up-

regulation of miR-16, detected by Northern analyses. The strongest up-regulation was

found in HEK293, probably because it had the highest transfection efficiency. These

data strongly suggest that miR-15a and miR-16-1 can be expressed through this splice

form of DLEU2. Since the promoter region and first exon of DLEU2 are deleted in the

10 kb minimal region of loss these data suggest that this small deletion could also be a

reason for loss of miR-15a and miR-16-1, in contrast to the study arguing for a 30 kb

minimal region of loss encompassing only the miRs (6).

The cell lines show different levels of endogenous miR-16 expression. MCF7 seems to

have high levels of endogenous expression compared to HEK293 and U2OS. However,

since the mature miR-16-1 has the same sequence as miR-16-2, which is located on

chromosome 3 (6), we cannot know for sure which of these that is detected in the mock

transfected control samples.

In the transfected samples the pre-microRNAs are detected since they include the

sequence of the mature miRNA. However, they are for some reason generally not seen

in the negative controls, not even in MCF7 that show a relatively high expression of the

mature miRNA. A possible explanation could be that they normally are degraded, and

that this degradation process has not occurred yet in the transfected cells. The levels of

the primary miRNA transcript are also a lot higher in the transfected cells.

The DMSO treatment of HL60 suggests that miR-16 is up-regulated after 24 h of 1%

DMSO treatment. This would be an interesting finding as it could mean that DLEU2 is

repressed by c-Myc. However, the control used in this experiment is not satisfactory

23

since it was harvested at time point 0, and we do not know if miR-16 levels would

increase in non-treated cells after 24h. This experiment should be repeated with proper

controls.

Method

The most common method today to analyze the expression of microRNAs is northern

blot analysis combined with polyacrylamide gels. However, this technique was not

sensitive enough to detect changes in microRNA expression in the siRNA experiment.

The HEK293 cells express rather low endogenous levels of miR-16 and to be able to

detect even lower levels a more sensitive technique must be used. There are several

ways to increase the sensitivity of the method. One way is to use locked nucleic acid

(LNA) probes that give much stronger hybridization (20). Solution hybridization and

Real Time PCR are other more sensitive techniques for detection of microRNAs.

Relevance

This study confirms that the microRNAs miR-15a and miR-16-1 can be expressed

through an alternative splice form of the candidate tumor suppressor gene DLEU2,

previously cloned and characterized by Liu et al (7), as a form termed DLEU2MIR,

where the miRs are included in a 3' exon. There are other examples described where

microRNAs are present in 3'-UTRs of non-protein coding RNA transcripts. The miR-

155 has been found to be encoded by BIC (21) and the gene C13orf25 can be

alternatively spliced to either include 8 microRNAs within its 3'-UTR or use a

downstream 3' exon thus excluding the microRNAs from the transcript (22), similar to

DLEU2.

The data from the present study gives further evidence that the microRNAs miR-15a

and miR-16-1 might be involved in the pathogenesis of CLL. The next important step is

to find target genes that are regulated by the microRNAs. There are bioinformatical

tools available using algorithms to identify potential binding sites for a given miRNA in

genomic sequences, for example miRBase Targets at the Sanger Institute website (23).

However, the understandings of in vivo miRNA target selection mechanisms are still

limited, and a miRBase target search for miR-15a and miR-16 gives over 500 hits.

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Bcl2 has been suggested as a target by Cimmino et al (16) and they also found a 9 bp

sequence in the 3'-UTR of Bcl2 that is complementary to both miR-15a and miR-16.

Recent Western blot data from our group confirm that Bcl2 translation is inhibited by

overexpression of DLEU2MIR.

Preliminary data from our group also show that Cyclin E levels were lowered in cells

transfected with DLEU2MIR. It has been shown that the absence of somatic mutations

in specific immunoglobulin heavy-chain variable region genes (IgVH) is associated with

ZAP70 expression and poor prognosis (14). ZAP70 is involved in cell cycle and

apoptosis regulation. Bogner et al showed that cyclin E is expressed significantly higher

in ZAP70 positive samples (25). High cyclin E may lead to increased proliferation of

the malignant cells. If miR-16 is involved in the regulation of cyclin E this would be an

important finding that could explain why loss of miR-16 could lead to tumor

development. Conserved 3'-UTR target sites for miR-16 are found in cyclin E (Figure

11).

25

Figure 11. Two conserved miR-16 target regions are found in the 3'-UTR of Cyclin E (CCNE1).

26

There are a number of interesting putative targets for miR-16, see table 1.

Gene name Description

CD40 Member of the TNF-receptor superfamily. This receptor has been found to be essential in mediating a broad variety of immune and inflammatory responses.

Bcl2 This gene encodes an integral outer mitochondrial membrane protein that blocks the apoptotic death of some cells such as lymphocytes.

Cyclin E This cyclin forms a complex with and functions as a regulatory subunit of CDK2, whose activity is required for cell cycle G1/S transition. Overexpression of this gene has been observed in many tumors, which results in chromosome instability, and thus may contribute to tumorigenesis.

Cyclin D1 This cyclin forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. Mutations, amplification and overexpression of this gene, which alters cell cycle progression, are observed frequently in a variety of tumors and may contribute to tumorigenesis.

Cyclin D2 This cyclin forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition. High level expression of this gene has been observed in tumors.

MYB The c-Myb protein is a transcription factor that regulates specific genes during development and cellular differentiation. The c-Myb protein has latent transforming activity that can be unleashed through point mutations and deletions, so minor changes can convert it to a potent transforming protein that induces leukemias in birds and rodents.

Table 1. Putative targets for miR-16.

The discovery that miR-15a and miR-16-1 can be expressed through a splice form of

the putative tumor suppressor gene DLEU2 is an important step in the characterization

of how 13q14 deletion can lead to tumor development. A better understanding of the

molecular genetics behind a disease is crucial for finding new therapeutic targets. The

microRNAs are interesting from this point of view, since these small molecules may be

relatively easy to interfere with or replace.

27

Acknowledgements

I wish to express my gratitude and appreciation to:

Martin Corcoran, my supervisor, for teaching me all the techniques and explaining the

complex background to this project.

Dan Grandér, head of the group, for all you help and support and for giving me the

opportunity to work with this exciting project.

Mikael, Lotte and the other members of the group for helping me with all sorts of lab

related problems.

And thank you Louise Forssell for having lunch with me on a regular basis, never

complaining over my complaints.

28

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