search of mirnas critical for medulloblastoma formation using mirage method
TRANSCRIPT
Search of miRNAs critical for medulloblastoma formation using MiRaGE method
○Y-h. Taguchi(Dept. Phys., Chuo Univ.)Jun Yasuda(Tohoku Univ.)
Present address:Cancer Research Inst. Ariake, Tokyo
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Experiments microRNA vs tumor
Target genes
Computer oriented prediction (uncertain)
genome
microRNA
microRNA
mRNA
mRNA
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miRNA1 ○ × ○ ○ ○ ○ × ×miRNA2 ○ × ○ ○ × × ○ ○miRNA3 × ○ ○ × ○ ○ × ×miRNA4 ○ ○ ○ × ○ ○ × ×
Gen
e1G
ene2
Gen
e3G
ene4
Gen
e5G
ene6
Gen
e7G
ene8
miRNA target gene list (計算機による予測)simple seed match
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Murine Medulloblastoma(MB)Murine Medulloblastoma(MB) (Dr. Tetsuo Noda’ group(Dr. Tetsuo Noda’ group
(The JFCR-Cancer Institute)).(The JFCR-Cancer Institute)).
gene1gene2gene3
予測
miRNA1
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Materials
P6P6=6 days after birth, normal but growingP30P30=30 days after birth, normal and not growingMBMB=a few month after birth, malignant neoplasm30% of the Ptc1 +/- mice suffers from MB.
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mRNA/miRNA expression byArray: AgilentatP6, P30 and MB
log(xg[mRNA/miRNA:MB or P6]) vs
log(xg[mRNA/miRNA:P30])
xg: mRNA/miRNA expression
Target gene list: simple seed match
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log(xg[miRNA:P6/MB]) vs log(miRNA:xg[P30])
of considered miRNA(*)
(*) each miRNA is measured by multiple probe
t te s t fo r m iRNA e x p re s s io n
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log(xg[mRNA:P6/MB]) – log(xg[mRNA:P30]) in target genes of considered miRNA
t test for miRNA target genes (MiRaGE method)
log(xg[mRNA:P6/MB]) – log(xg[mRNA:P30]) in target genes of
any of other miRNA
VS
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P30 P6/MB
Gether the information of miRNA targets
Compare the expressions of targets for each miRNAs
Calculate False Discovery Rate
Generate ranking
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miRNA Targets Down P-value FDR
miR-a 54 3 0.5 0.4
miR-b 120 54 0.0001 0.005
miR-c 36 1 0.5 0.7
... ... ... ... ...
miR-X 60 18 0.001 0.007
Reject miR-a & c because the FDR > 0.05
Filtrate with miRNA expression profiles
Ranking
MiRaGEMiRaGE
1 1
1 mmu-miR-25mmu-miR-25 1 1
2 mmu-miR-466i-5p 1 1
3 mmu-miR-92ammu-miR-92a 0.75 1
4 mmu-miR-19ammu-miR-19a 1 0.69
5 mmu-miR-19bmmu-miR-19b 1 0.69
6 mmu-miR-3082-5p 1 0.56
7 mmu-miR-130a 1 0.5
8 mmu-miR-130b 1 0.5
9 mmu-miR-15b 1 0.5
10 mmu-miR-2861 1 0.5
11 mmu-miR-3096-5p 1 0.5
12 mmu-miR-32 0.5 1
13 mmu-miR-322 1 0.5
14 mmu-miR-721 1 0.5
15 mmu-miR-149* 0.5 0.88
16 mmu-miR-3081* 1 0.38
17 mmu-miR-574-5p 1 0.31
18 mmu-miR-669n 0.5 0.81
19 mmu-miR-1187 1 0.25
20 mmu-miR-182mmu-miR-182 0.5 0.75
miRNA P30<<MBtarget gene P30>>MB
miR-17~92 cluster family miR-17~92 cluster family membersmembers are ranked in top 5 by combination of MiRaGE methods and miRNA expression profiling.
selected bymiRNA miRNA expression / MiRaGE
suggested contribution suggested contribution to cancer formationto cancer formation
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mmu-miR-100 1 1
mmu-miR-126-3p 1 1
mmu-miR-29cmmu-miR-29c 1 1
mmu-miR-376ammu-miR-376a 1 1
mmu-miR-451 1 1
mmu-miR-99b 1 1
mmu-miR-136* 1 0.9375
mmu-miR-299* 0.75 1
mmu-miR-26ammu-miR-26a 1 0.5
mmu-miR-26bmmu-miR-26b 1 0.5
mmu-miR-29ammu-miR-29a 0.5 1
mmu-miR-7a-1*mmu-miR-7a-1* 1 0.5
mmu-miR-3107 1 0.4375
mmu-miR-340-5p 1 0.3125
mmu-miR-369-5p 1 0.3125
mmu-let-7ammu-let-7a 1 0.25
mmu-let-7emmu-let-7e 1 0.25
mmu-let-7gmmu-let-7g 1 0.25
mmu-let-7immu-let-7i 1 0.25
mmu-miR-467b 0.25 1
selected bymiRNA miRNA expression / MiRaGE
tumor-suppressive miRNAs tumor-suppressive miRNAs
neuron-specific miRNAsneuron-specific miRNAs
miRNA P30>>MBtarget gene P30<<MB
Some of the neuron-neuron-specific miRNAspecific miRNAs and tumor-suppressive tumor-suppressive miRNAsmiRNAs seem to contribute to the gene expression profiles of P30.
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miRNA P30<<P6target gene P30>>P6
miR-17~92, miR-17~92, mir-106b-25mir-106b-25 ,,mir-106a-363mir-106a-363cluster family members are ranked in top 5 by combination of MiRaGE methods and miRNA expression profiling.
selected bymiRNA miRNA expression / MiRaGE
1 mmu-miR-106bmmu-miR-106b 1.00 1.00
2 mmu-miR-130a 1.00 1.00
3 mmu-miR-130b 1.00 1.00
4 mmu-miR-15b 1.00 1.00
5 mmu-miR-17mmu-miR-17 1.00 1.00
6 mmu-miR-20ammu-miR-20a 1.00 1.00
7 mmu-miR-20bmmu-miR-20b 1.00 1.00
8 mmu-miR-301b 1.00 1.00
9 mmu-miR-322 1.00 1.00
10 mmu-miR-721 1.00 1.00
11 mmu-miR-93mmu-miR-93 1.00 1.00
12 mmu-miR-542-3p 1.00 0.94
13 mmu-miR-3081* 1.00 0.88
14 mmu-miR-335-3p 1.00 0.88
15 mmu-miR-199a-5p 1.00 0.81
16 mmu-miR-199b* 1.00 0.81
17 mmu-miR-19ammu-miR-19a 1.00 0.81
18 mmmmu-u-mmiiRR--1199bb 1.00 0.81
19 mmu-miR-148a 0.75 0.94
20 mmu-miR-214 1.00 0.63
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selected bymiRNA miRNA expression / MiRaGE
tumor-suppressive miRNAs tumor-suppressive miRNAs
neuron-specific miRNAsneuron-specific miRNAs
miRNA P30>>P6target gene P30<<P6
Some of the neuron-neuron-specific miRNAspecific miRNAs and tumor-suppressive tumor-suppressive miRNAsmiRNAs seem to contribute to the gene expression profiles of P30.
mmu-miR-29c 1.00 1.00
mmu-miR-376ammu-miR-376a 1.00 1.00
mmu-miR-451 1.00 1.00
mmu-let-7bmmu-let-7b 1.00 0.94
mmu-let-7emmu-let-7e 1.00 0.94
mmu-let-7gmmu-let-7g 1.00 0.94
mmu-let-7immu-let-7i 1.00 0.94
mmu-miR-98 1.00 0.94
mmu-miR-126-3p 0.75 1.00
mmu-miR-299* 0.75 1.00
mmu-miR-29a 0.75 1.00
mmu-let-7ammu-let-7a 0.75 0.94
mmu-miR-3070b-3p 1.00 0.69
mmu-miR-138 1.00 0.63
mmu-miR-3107 1.00 0.56
mmu-miR-181a-1* 0.50 1.00
mmu-let-7dmmu-let-7d 0.50 0.94
mmu-miR-1937b 0.25 1.00
mmu-miR-1937c 0.25 1.00
mmu-miR-337-5p 1.00 0.25
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MiRaGE method + miRNA expression successfully pick up biologically important miRNAs. Further (wet) experiments which supress miRNA expression with tiny LNA is now planed.
If it is successful, our method can find miRNAs which control tumor formation.
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Significance of reciprocal relationship between miRNA and its target genes.
t.test of P-values between top n miRNAs and others:
P30 → MBP(mRNA:down|miRNA:up)P(mRNA:up|miRNA:down) P(miRNA:down|mRNA:up) P(miRNA:up|mRNA:down)
P=0.05
⟨ log10P⟩
n
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Significance of reciprocal relationship between miRNA and its target genes.
t.test of P-values between top n miRNAs and others:
P30 → P6P(mRNA:down|miRNA:up)P(mRNA:up|miRNA:down) P(miRNA:down|mRNA:up) P(miRNA:up|mRNA:down)
P=0.05
⟨ log10P⟩
n
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MiRaGE method + miRNA expression satisfy reciprocal relationship very well. In our knowledge, this is for the first time to do this for such a large number of miRNAs
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Discussion: What causes successful achievement?
Point 1:Usage of “good” maicroarryAffymetric ☓Agilent ○
Point 2:Negative set = genes not targeted by considered miRNA but done by other miRNAs
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As for Points:Although we do not know the reason, off-target genes targeted by other miRNAs are more expressive.
let-7a transfection (Taguchi & Yasuda, 2010)
off target target
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Conclusion:
MiRaGE (MiRNA Ranking by Gene Expression) method is very simple, but
1) can successfully pickup biologically important genes
and
2) can detect reciprocal relationship between miRNAs and their target genes (mRNA).
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Acknowledgements:Acknowledgements: We thank Drs. Tetsuo Noda We thank Drs. Tetsuo Noda and Katsuyuki Yaginuma for and Katsuyuki Yaginuma for
providing reagents.providing reagents.These works were supported These works were supported by KAKENHI (23300357) .by KAKENHI (23300357) .
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