“Validation and biological characterization of new microRNAs on carcinogenic process”

Ma. Catalina Güida

Functional Genomic Laboratory. Cancer Program Institut Pasteur de Montevideo.

Journal ClubApril 15, 2009.

Institut Pasteur of Montevideo

New miRNA cloned from B-Chronic Lymphocytic Leukemia (B-CLL)

Dalmay T, J.Int Med 2008

miRNAs biosynthesis

Dalmay T, J.Int Med 2008

miRNAs Activity

B-Chronic Lymphocytic Leukemia (CLL)

The commonest leukemia of Western countries.

Characterized by elevated numbers of circulating clonal leukemic B cells.

Shows variable clinical course.

Biological markers: - mutational status of the immunoglobulin heavy-chain variable region-gene (IgVH) - the expression levels of the ZAP-70 tyrosine kinase - the expression of CD38+ - presence of cytogenetic abormalities (11q or 17p deletions).

NM: unmutated IgVH, or with hight expression of ZAP-70 or CD38 and/or 11q or 17p deletion rapidly fatal course.

M: mutated clones or low ZAP-70 or CD38 expression and no deleterious cytogenetic abnormalities indolent course and frequently die by unrelated diseases.

Putative targets for the news miRNAS

Possible Tumor Suppressor genes in 1p36.31

Baghi, et.al. Cell (2007)

Putative targets for the news miRNAS

CHD Family Proteins

J. Adam Hall and Philippe T. Georgel Biochem. Cell biol. 85: 463-476 (2007)

Model for Chd5 in Tumor Suppression

Identify a novel tumorigenic pathway which could be activated through a microRNA-induced deficiency of the chromatin-remodeling protein CHD5 expression.

This could predispose to malignancy by crippling tumor suppressive pathways involving p16lnk4a, p19Arf and p53. We will try to establish an unrecognized role of hCHD5 in both facilitating transcriptional programs providing tumor suppression and as an alternative regulating pathway for the p19Arf/Mdm2/p53 axis.

Major Objectives

Specific Objectives

Validation of human CHD5 as a target of novel microRNAs miR-1201, miR1202 and miR1203 (http://microrna.sanger.ac.uk/ sequences/index.shtml).

Determination of p53-dependent and/or p53-independent pathways for the oncogenic microRNAs (miR-1201, miR1202 and miR1203)

Effects of miRNA-dependent modulation of CHD5 levels on cell cycle, apoptosis, senescence and proliferative activity of tumoral cell lines.

Analysis of oncogenic microRNAs in human hematological and neurological malignancies in order to determine their potential as therapeutic targets

Study of biological function of miRNA

Phenotypic characterization (Oncogenic properties)

Target validation

Bioinformatic prediction algorithms

Reporter assay

In situ hybridizations

Overexpression and silencing technologies

Design Sense and Antisense of miRNA- 1201, 1202 and 1203 to be transfected into the cells (Overexpression and silencing technologies).

Some tools

Clone 3’ UTR of the putative target into a Renilla vector (Reporter assay).

miRNAs Sense and Antisense

miR- 1201 S 5’ CCU GAU UAA ACA CAU GCU CUG A 3’AS 5’ U CAG AGC AUG UGU UUA AUC AGG 3’  mC*CmU*GmA*mUmUmAmAmAmCmAmCmAmUmGmCmUmC*mU*mG*mA/3Cy3Sp/mU*mC*mA*mGmAmGmCmAmUmGmUmGmUmUmUmAmAmUmC*mA*mG*mG/3Cy3Sp/

Oligo Base Types

2’ O-Methyl Ribonucleotide bases (20)

Modifications

Phosphorothioate Bond (6) (*)3’ Cy3TM-Sp (1)

Transfeccion of miRNA in SK-N-SH

1201 sense 10 nM 16 hs(Lipofectamine 200)

SK-N-SH

Merge

Experimental model

Neuroblastome cell lines without 1p36 deletion: SKN.SH IMR32

CHD5 mRNA expresionChd5 protein expresionmiRNA 1201,1202 and 1203 expresion levels

E10 (207 pb) E11 (212 pb) E12 (132 pb) E13 (109 pb) E14 (192 pb)

CHD5 Gen

Set 2

Set 1 Set 3

mRNA CHD5 and novel miRNAs characterization on neuroblastoma cell lines

SK-N-SH transfected with AS miRNAs

M2

M3

M4 M5

M2

M3

M4 M5

M2

M3

M4 M5

M2

M3

M4 M5

Control

AS 1203AS 1202

AS 1201

2.59

5.34

11.86

5.35

FACS Analysis

SK-N-SH transfected with AS miRNAs

24 hs post-transfection

KD= Know down of miRNAs ( target genes )

KD= Knock down of miRNAs ( target genes )

Difficulties and troubles CHD5 Protein detection:

N terminal C terminal aa residues Antibody

1395 1407 13 ANtiCHD5 from Everest, Novus Biologicals

1521 1705 185 AntiCHD5 from Santa Cruz

1550 1602 101 AntiCHD5 from Abcam

1603 1702 100 AntiCHD5 from Strategic Diagnostic

CHD5 ORF

Looking for an alternative system

- 250 kD- 170 kD

Tubulin

HCT116

HFF

MEF -/

-

WI3

8H12

99

DU.145

MCF7

U2O5

MEF +/+OVCA.3

Chd5/Gapdh

Transfection of WI38 with sense and antisenseof miRNAS

KD= Knock down of miRNAs ( CHD5 )OE= Over Expression of miRNAs ( CHD5)

WI3

8KD

Control

OE

Control

Gapdh

p53

Chd5

ControlControl Control

WI38 transfected with separate and pooled miRNA

KD= Knock down of miRNAs ( target genes )OE= Over Expression of miRNAs ( target genes)

170kDa

130kDa

100kDa

70kDa

250kDa

p53

Gapdh

1203 12011202 1203 1202 1201PoolASControl

Pool S

AS S

WI38 - 48 hs post transfection

170kDa

130kDa

100kDa

70kDa

170kDa

130kDa

100kDa

70kDa

250kDa

10 nM20 nM10 nM20 nM10 nM20 nMsiControl sip53 siCHD5

p53

Gapdh

E10 (207 pb) E11 (212 pb) E12 (132 pb) E13 (109 pb) E14 (192 pb)

CHD5 Gen

Set 2

Set 1 Set 3

qRT-PCR with 3 set of primers for CHD5

Northern blot assay

Mouse B

rain

Jurka

t

Daudi

SK-N-S

H

SK-N-B

E2

Co-transfection with Renilla vector with 3’ UTR CHD5 and sense of miRNAs 1201-1202-1203

Cloning of 3’UTR of target genes in the 3’UTRof reporter gen vector

Brennecke, et al. PLoS Biology, 2005

miRNA-mRNA interaction

miRanda v3.0&

TargetScanmicroRNA Target Scanning Algorithm

Position 1863-1869 of CHD5 3' UTR 5' ...GCUCUGCGGUGCCUCCUGGCAAA...

                       ||||||  

hsa-miR-1202 3'     GAGGGGGUGACGUCGACCGUG

   

Position 3020-3026 of CHD5 3' UTR 5'    ...UGGGUGGGGGCCGAGGCUGGCAC...

                          ||||||| 

hsa-miR-1202 3'         GAGGGGGUGACGUCGACCGUG

   

Position 3448-3454 of CHD5 3' UTR 5' ...CAGGCACAGCCCCAG----GCUGGCAA...

                 |||||     ||||||| 

hsa-miR-1202 3'          GAGGGGGUGACGUCGACCGUG

   

Position 1017-1023 of CHD5 3' UTR 5'    ...GCCUACCCUGCCCACCUCCGGAG...

                          ||||||  

hsa-miR-1203 3'        CUCGACGUAGGACCGAGGCCC

   

Position 2087-2093 of CHD5 3' UTR 5'        ...CUGUGCGCCUUCCUCUCAGGCAG...

                              ||||||  

hsa-miR-1201 3'         AGUCUCGUACACAAAUUAGUCCGA

   

   

position 1314  

1201 

CHD5 3’UTR - 3681 pb

1201

1203

12021201

1202

1202

12011203

12011202

1202 1202

1st part -3’UTR CHD5

2nd part -3’UTR CHD5

3th part -3’UTR CHD5

832 pb

693 pb

698 pb

Cloning strategy

Conclusions

The AS transfection showed a biological effect in neuroblastoma cell line, suggesting that these miRNA could have oncogenic properties.

CHD5: According the western blot, real time RT-PCR we can not confirm yet that CHD5 gen is regulated by the new miRNAs.

Perspectives

Difine conditions for detection of CHD5 protein.

siCHD5 assays

Same assays with Prdm16 and Hes 3’UTR (Luciferasa, miRNAS OE and KD, siARN).

Phenotype characterization of cells with sense and antisense of 1201, 1202 and 1203 miRNAs.

Characterization of this miRNAs and their targets in B-LLC

Thank you!

Alfonso Cayota

Ma. Rosa GarcíaJ. Pablo Tosar

Moshe Oren

Yael Aylon

Florencia Cabrera

Alvaro Pena

Julia Sanguinietti

Fernanda Bangueses

Braulio Bonilla

CHD5 in neuroblastoma cell lines

Chd5/Gapdh

IM

R-3

2

IM

R-3

2

Jurk

at

250 kD

95kD

75 kD

Rat Brain

250 kD

95 kD

1201-1202-1203 miRNA levels in different cell lines

WI38

Transfection with sense of miRNA 1201-1202-1203 (Over Expression)

Transfection with antisense of miRNA 1201-1202-1203 (Knock-down)

SK-N-SH

Transfection with antisense of miRNA 1201-1202-1203 (Knock-down)

Transfection of WI38 with sense and antisenseof miRNAS

Transfection of WI38 with senseof miRNAS and siCHD5

Gapdh

p53

170 kD -- -- 170 kD -- 250 kD

OEsiC

HD5

Contro

l -

OE

OE= Over Expression of miRNAs ( CHD5)

WI38 - 48 hs post transfection

WI38 transfected with separate and pooled miRNA

KD= Knock down of miRNAs ( CHD5 )OE= Over Expression of miRNAs ( CHD5)

OE KD OE KD

KD= Knock down of miRNAs ( target genes )OE= Over Expression of miRNAs ( target genes)