MicroRNAs can function as tumour suppressor and oncogenes

MicroRNA are subject to genomic regulation

MicroRNA are subject to transcriptional and post-trascriptional regulation

RNA expression patterns dictate miRNA repressibility in cells

Mechanisms underlying the global downregulation of miRNAs in cancer

Changes in miRNA biogenesis.

Transcriptional repression by oncogenic transcription factors (MYC oncoprotein directly contributes to the global transcriptional silencing of miRNAs).

Cancer cells present global downregulation of miRNAs, loss of tumour-suppressor miRNAs and specific accumulation of oncogenic miRNAs.

AGO, Argonaute; DGCR8, syndrome critical region 8; E2, 17oestradiol; ER, oestrogen receptor-; HNRNPA1, heterogeneous nuclear ribonucleoprotein A1; KHSRP, KHtype splicing regulatory protein; m7G, 7methyl-guanosine; p53mt, mutant p53; p53wt, wild-type p53; SNIP1, SMAD nuclear interacting protein 1; SRSF1, serine/arginine-rich splicing factor 1; TARBP2, transactivation responsive RNA-binding protein 2; XPO5, exportin 5.

Regulation of miRNA biogenesis pathway by processing factors

Mechanisms of miRNA perturbation in cancer

RISC-associated factors regulate efficient microRNA-mediated repression

Contribution of miRNAs to cancer pathways

Contribution of miRNAs to cancer pathways

Contribution of miRNAs to cancer pathways

Contribution of miRNAs to metastasis

A microRNA expression signature of human tumors

MicroRNAs as diagnostic and prognostic tools

Clustering of miRNA expression in different ALL subtypes

MicroRNAs as diagnostic and prognostic tools

MicroRNAs therapeutic tools

Re-Expression of miRNAs

I. Synthetic small RNAs called miRNA mimetics (exact sequence of the endogenous miRNA)

II. DNA vectors (miRNAs precursors sequences, degradation) III. Viral vectors

Anti-miRNAs

I. Antisense oligonucleotides that bind directly to miRNAs II. AntagomiR III. miRNA sponges

Target Protectors (small oligonucleotides with perfect complementary to the seed region and to the 5 and 3 flanking sequences in the 3UTR of specific mRNA target)

MicroRNA Silencing in Primates: Towards Development of Novel Therapeutics

Antisense oligonucleotides

They work by stoichiometric interaction with mature miRNAs, either titrating them or binding to miRNA precursors and inhibiting the biogenesis of mature miRNA

1. 2O-methyl RNA oligonucleotides, 2O-methyl group increases stability

2. 2-O-methoxyethyl oligonucleotides (2-MOEs)

3. 2,4-methylene bridged nucleic acids (locked nucleic acid, LNA)

In vivo knockdown of miRNAs

Silencing of microRNAs in vivo with antagomirs

The antagomir is a 2O-methylated oligonucleotide with a cholesterole molecule linked at its 5 end that improve the cellular uptake of this molecule.

liver

Lentivirus-mediated antagomir expression

Design of U6 sponges by subcloning the microRNA binding site region into a vector containing a U6

Design of microRNA sponge by inserting multiple binding site into the 3 UTR of GFP reporter gene driven by the CMV promoter

Sponge with bulged binding sites were designed to protect duplex against endonucleolytic cleavage by AGO2

In vivo miRNA expression

In vivo miRNA inhibition

NATURE PROTOCOLS

An integrated biological knowledge and analytic tools aimed at systematically extracting biological meaning from large protein/gene lists

http://david.abcc.ncifcrf.gov

PROCEDURE 1) Submit a gene list to DAVID: -Copy and paste a list of gene Ids into Box A - Select the appropriate gene identifier type - Click the Submit List buttom

2) Access DAVID analytic modules

Functional annotation chart Pathway map viewer. The red star indicates the associations between pathway genes and the users input genes. Following the pathway flow, IL10 was activated as an upstream immune stimulator. Then, the middle stream gene, HO-1, was involved. IL-1/TNF/IL-6, as downstream regulator, was finally activated. Thus, the users genes may be analyzed in a network context.

RESULTS

Proposed scheme for the treatment of liver cancer with combined chemotherapy and miRNA-based therapy

Role and mechanism of polycistron mir-143 mir-145

in regulation of CRC progression

miR-143 and miR-145 genes map to a 2Kb region on chromosome 5 and they are transcribed in the same polycistron as a single pri-miR.

miR-143 and miR-145 are down modulated in colon cancer and other tumoral tissues such as lung, breast and lymphoma (Akao et al., 2007).

We study miR-143 and miR-145 in an integrated framework of common targets or complementary pathways and not

such as a single miR/single target

miR-143 and miR-145

Experimental plan

Proliferation Migration and colony formation assay

Chemosensitivity Tumorigenic potential in vivo

Functional assays

Target analysis

Protein modulation miR-143 and miR-145 reporter assay

We have investigated the anti-proliferative effect of mir-143 and mir-145 in colon cancer cell lines after their overexpression.

Rescue assays

Functional assays: proliferation

Upon prolonged culturing the two populations (Tw and Tw143-5) became indistinguishable in terms of growth proprieties

TWG

TWG 143-5

5LTR CMV EGFP PGK 3LTR

5LTR CMV EGFP PGK 3LTR pri miR143-5

1

10

100

1000

3 4 6

Rel

ativ

e m

iRN

A le

vels

[T

w 1

43-5

/Tw

]

days

miR143 miR145

0

10000

20000

30000

40000

50000

60000

0 1 3 4 6 C

ells

Num

ber

days

TW TW 143-5

tTR

tTR

KRabTetR

Repression

Expression Noexpression

Noexpression

Tet on-off system

Tet on-off system for reversible induction of miR-143 and miR-145

rtTA

tTR

+ Puromycin + Neomycin

LTR or

LTR 143-5

Sw480 cell

lines

Virus infection

Virus infection Sw480

cell lines

+ Doxycyclin

RFP-positive cells are sorted

pLV-rtTA

pLV-tTR

5LTR SV40 tTR TK 3LTR NEO

5LTR CMV PURO PGK 3LTR rtTA

pLTR

5 LTR RFP TRE 3LTR

pLTR143-5

Pri miR143-5 5 LTR RFP TRE 3LTR

Functional assays: proliferation

miR-143 and miR-145 inducible expression inhibit growth in colon cancer cell lines

0

10000

20000

30000

40000

DAY 0 DAY3 DAY4 DAY5

Cel

ls N

umbe

r

LTR

LTR143-5

1

10

100

1000

10000

DAY 3 DAY 6

Rel

ativ

e m

iRN

A le

vels

[L

TR 1

43-5

/LTR

]

miR143

miR145

Functional assay: migration and colony formation

SW480

LTR 143/5 LTR

LTR 143/5 LTR

SW480

miR-143 and miR-145 inducible expression reduce migration and anchorage-independent growth capabilities in colon cancer cell lines

0 20 40 60 80

100 120

48 h Num

ber o

f mig

rate

d ce

lls

LTR LTR 143/5

0

20

40

60

80

100

16 days

Num

ber o

f col

onie

s

LTR LTR 143/5

*

*

p < 0.05

p < 0.05

Functional assay: cytotoxicity

miR143 and miR145 overexpression increase sensibility to chemotherapeutic drug treatment.

0

20

40

60

80

100

120

25uM 50uM 100uM 5uM 10uM 20uM 5uM 10uM 20uM

5FU IRINOTECAN OXALIPLATIN

% c

ell v

iabi

lity

LTR LTR 143-5

** ** **

** p < 0.01

0 0,2 0,4 0,6 0,8

1 1,2 1,4 1,6

3 4 5 6 7

Volu

me

(cm

3)

weeks

TW TW143-5

Functional assay: tumorigenic potential in vivo

73%

TW

GFP

HLA

89%

TW 143-5

GFP

HLA

miR-143 and miR-145 overexpression delay tumor

growth in mice

0

1

2

3

4

5

6

Rel

ativ

e m

iRN

A le

vels

[T

W 1

43-5

/TW

]

miR143 miR145

Target analysis

Target of miR- 143

Target of miR- 145

KRas, BRaf , KLF5, CD44, ErbB3

KLF5, CD44, ErbB3

p53

CD44

Genotoxic stress

c-myc

apoptosis cytostasis

miR-145 miR-143

Migration invasiveness

survival

?

KLF5

Target analysis : protein modulation

Klf5, Raf, Ras and CD44 protein levels decrease in presence of

miR143 and miR-145 0

20

40

60

80

100

120

TW TW 143-5

% o

f Erb

B3

prot

ein

leve

ls

0

20

40

60

80

100

120

LTR LTR 143-5

% o

f CD

44 p

rote

in

leve

ls

Ras

Raf

klf5

-actin

20 KDa

62 kDa

51 kDa

miR143-5

+ _

miR-143 and miR-145 target site

Gene Position Target Site

KRAS 1602-1608 5' ...UCAUGUUAAAAGAAGUCAUCUCA... (3UTR) ||||||| 3' CUCGAUGUCACGAAGUAGAGU (miR-143)

KRAS 3647-3653 5' ...ACAGUUUGCACAAGU--UCAUCUCA... (3UTR) ||| ||||||| 3' CUCGAUGUCACGAAGUAGAGU (miR-143)

KLF5 112-118 5' ...GAAAACCACAACUAAAACUGGAA... (3UTR) ||||||| 3' UCCCUAAGGACCCUUUUGACCUG (miR-145)

KLF5 238-244 5' ...UUACUCAAGCAGAUC-UCAUCUCA... (3UTR) ||| ||||||| 3' CUCGAUGUCACGAAGUAGAGU (miR-143)

CD44 784-790 5' ...CUUCUA-AGU-CUUCAUCUCA ... (3UTR) ||| ||| ||||||||| 3' CUCGAUGUCACGAAGUAGAGU (miR-143)

CD44 3387-3393 5' ... UUUUCAACUUGAAAGAAACUGGAC... (3UTR) ||||||| 3' UCCCUAAGGACCCUUUUGACCUG (miR-145)

BRAF 459-465 5' ... CUUUCAGUGCUACCUUCAUCUCU... (CDS) ||||||| 3' CUCGAUGUCACGAAGUAGAGU (miR-143)

0

0,2

0,4

0,6

0,8

1

1,2

1,4

143 145 143a 143b 143 145 143

Klf5 Ras CD44 Raf

Rel

ativ

e R

enill

a Le

vels

(wt/m

ut)

* * * * *

Luc SV40 TK Ren PolyA 3UTR/CDS wt or mut

miRNA

Reporter assay

Kllf5, Ras and CD44 genes are direct target of miR-143 and miR-145

* p < 0.05

Rescue assay: proliferation

0

10000

20000

30000

40000

0 2 3 4

cell

num

ber

Days

LTR

LTR143-5

LTR143-5 TW-RAS

0

10000

20000

30000

40000

0 1 2 3 4

cell

num

ber

Days

LTR

LTR143-5

LTR143-5 TW-RAF

0

10000

20000

30000

40000

0 1 2 3 4

cell

num

ber

Days

LTR

LTR143-5

LTR143-5 TW-KLF5

0

10000

20000

30000

40000

50000

60000

0 1 2 3 4

cell

num

ber

Days

LTR

LTR143-5

LTR143-5 TW-CD44

Target gene restoration induced an increase in SW480 proliferation

Rescue assay: migration

0

20

40

60

80

100

120

140

160

LTR LTR 143-5 LTR 143-5 Ras LTR 143-5 RAF LTR 143-5 KLF5 LTR 143-5 CD44

% o

f mig

rate

d ce

lls

*

*

* * *

Target gene restoration induced an increase in SW480 migration ability

* p < 0.05

Rescue assay: proliferation and migration

0

10000

20000

30000

40000

50000

60000

0 1 2 3 4

cell

num

ber

Days

LTR

LTR143-5

LTR143-5 TW-KLF5 TW-RAS

0

20

40

60

80

100

120

LTR LTR 143-5 LTR 143-6 RAS KLF5

% o

f mig

rate

d ce

lls

+

** p < 0.01

Conclusion Overexpression of miR-143 and miR-145 in colorectal cell lines induced :

proliferation

migration

anchorage independent growth

chemosensitivity

tumorigenic potential in vivo

K-Ras, B-Raf, Klf5 and CD44 are targets of the miR-143 and miR-145.

Coexpressed miRs can share common targets that belong to the same or to strictly connected pathways.