Inference of target gene regulation via miRNAs during cell senescence by MiRaGE Server

Y-h. TaguchiDepartment of Physics

Chuo University

1. What is cell senescence?

2. What is miRNA?

3. Previous works (wet)

4. MiRaGE Method

5. Correlation between target gene regulation by miRNA and miRNA expression change during cell senescence

6. Summary & Conclusion

The cell division cannot continue forever for incubated cell lines. It must stop after several rounds of proliferation.

⇓This is called “cell senescence”, which is believed to be related to agingaging.

1. What is cell senescence?

Thus, cell senescence is caused by the interruption of cell divisions, typically by cell cycle arrests.

2. What is miRNA?

miRNA is a kind of non-coding RNA. miRNAs are believed to suppress target gene expression by degradation of mRNAs. Important features:

・ Typically, there are hundreds kinds of miRNAs found for each species (c.a., 1000 for human).≧

・ Each miRNA targets more than hundreds of genes. ・ miRNA mainly contributes to cell type change

(e.g., cancer, defferentiation, diseases) ・Infulence to target gene expression by miRNA is subtle (〜10%) and contexts dependent.・In spite of that, miRNA critically contributes to the related processesmiRNA critically contributes to the related processes (e.g., induction of cell cycle arrest)

3. Previous works (wet)

Several researches suggest the contribution of miRNAs to cell senescence.

Upregulation of miRNAs during cell senescencemiR-34a,miR-486-5p,miR-494,miR-210...

Downregulation of miRNAs during cell senescencemiR-15a/b,miR-20a,miR-92,miR-16b....

Induction of cell senescence by suppression of miRNA downregulated during cell senescence.

It is likely true that miRNAs contribute to cell senescence.

However, which one?

Dhahbi et al (2011) recently reported the upregulaton of

141(!) miRNAs and the downregulation of 131(!!) miRNAs during cell senescence by deep sequencing.

The reason why limited number of miRNAs revealed significant expression change during cell senescence seems to be due to less sensitivity of microarray analysis.Is it truly critical the down/upregulation of such large number of miRNAs for cell senescence?

4. MiRaGE Method

In order to select “Critical ” miRNAs during cell senescence, regulation of target genes by miRNAs is inferred by MiRaGE Server.

For details, see

SIGBIO-25-5: Search of miRNAs critical for medulloblastoma formation using MiRaGE method○Y-h. Taguchi(Chuo Univ.) ， Jun Yasuda(Tohoku Univ.)

SIGBIO-25-30:Gene expression regulation during differentiation from murine ES cells due to microRNA○Masato Yoshizawa，Y-h. Taguchi（Chuo Univ.）

significantly up/downregulated?

(t test, Wilcoxon test, KS test)

MiRaGE :MiRNA Ranking by Gene Expression

miRNA

target gene

target gene

VS

gene

considered miRNA

5. Correlation between target gene regulation by miRNA and miRNA expression change during cell senescenceA) Confirmation of independence of cell line

Two Cell Lines:IMR90 : young (PD 30) vs senescent (PD 48) vsMRC5 : young (PDL 28) vs senescent (PDL 63)

mRNA expression change (during cell senescence)

MiRaGE Server⇓

P-values attributed to each miRNA

Intersection between N top-ranked significant miRNAs based upon P-values (t test) IMR90 vs MRC5

downregulation

upregulation

10

10

30%

100%

20%

100%

500 1500N 500 1500N0

4

% o

f com

mo

n miR

NA

s

random

random

P=0.05

binomial: -log

10 P

P=0.05Thus, the results are cell line independent (possibly robust)

miRDeep2:genome alignment program for miRNA-seq

B) Confirmation of reciprocal relationship between miRNA expression change and P­value (upregulation of target genes)

Co

rrela

tion C

oe

fficient

# o

f miR

NA

s-log

10 P

quality score(miRDeep2)

1 102 104

2

1

quality score quality score

100

700

0.05

0.35

Threshold Value

IMR90,NGS

P=0.050

Candidates of miRNAs downregulaed(target genes are upreglated)

NMRC : Normalied miRNA Read CountsSCORE : miRDeeps scoreP-value : upregulation of target genesRFC : Reciprocal Fold Change : young/senescent

P<0.05 RFC>1.0

P<0.05 FC>1.0NMRC : Normalied miRNA Read CountsSCORE : miRDeeps scoreP-value : downregulation of target genesFC : Fold Change : senescent/young

Candidates of miRNAs upregulaed(target genes are downreglated)

FC>1.0P<0.05NMRC : Normalied miRNA Read CountsSCORE : miRDeeps scoreP-value : downregulation of targe genesFC : Fold Change : senescent/young

Candidates of miRNAs upregulaed(target genes are downreglated) continued

6. Summary & Conclusion

1. Selection of miRNAs commonly up/downregulated during cell senescence 2. Reciprocal relationship between target gene regulation and miRNA expression change

3. Reduction of number of critical candidate miRNAs during cell senescence (down: 131 ⇒１０, up: 141 32)⇒