Background and Aims of study

Take home notes• miR-1 and miR-133a expression were down-regulated in tumor tissues and their

expression were positively correlated in normal and tumor tissues.• Genome-wide analysis revealed that TAGLN2 and PNP were common target genes

of miR-1 and miR-133a.• TAGLN2 and PNP expression were inversely correlated with miR-1 and miR-133a

expression in HNSCC.• Loss-of-function assay indicates that TAGLN2 and PNP may act as oncogenes in

HNSCC.• TAGLN2 is a member of the calponin family of actin-binding proteins.

Overexpression of TAGLN2 was observed in hepatocellular carcinoma, lung adenocarcinoma, pancreatic and bladder cancer.

• PNP is an enzyme involved in purine metabolism. PNP catalyzes the conversion of adenosine to adenine, inosine to hypoxanthine, and guanosine to guanine, creating ribose phosphate in each case. PNP is considered a therapeutic target in malignant lymphoproliferative diseases.

Molecular networks regulated by tumor suppressive microRNA-1 and microRNA-133a in head and neck squamous cell carcinomaAbstract #3152

Nijiro Nohata1,2, Toyoyuki Hanazawa1, Takashi Kinoshita1,2, Naoko Kikkawa1, Miki Fuse2, Takeshi Chiyomaru3, Hirofumi Yoshino3, Hideki Enokida3, Masayuki Nakagawa3, Yoshitaka Okamoto1 and Naohiko Seki2

1Department of Otorhinolaryngology / Head and Neck Surgery, Graduate School of Medicine, Chiba University, Chiba Japan2Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan3Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan

AACR 103rd ANNUAL MEETING, Tuesday April 3, 2012, 8:00 AM – 12:00 PM

No. Symbol Gene nameLog2 ratio

miR-1 site miR-133a sitemiR-1 miR-133a Average

1 TAGLN2 transgelin 2 -2.78 -1.98 -2.38 3 2

2 PVRL2 poliovirus receptor-related 2 (herpesvirus entry mediator B) -2.7 -1.9 -2.3 - -

3 C4orf34 chromosome 4 open reading frame 34 -3.01 -1.51 -2.26 2 1

4 KATNAL2 katanin p60 subunit A-like 2 -2.65 -1.69 -2.17 - -

5 CGA glycoprotein hormones, alpha polypeptide -2.38 -1.92 -2.15 - -

6 TCTEX1D2 Tctex1 domain containing 2 -2.12 -2.14 -2.13 - -

7 hCG_1817306 hypothetical LOC100130691 -1.79 -2.22 -2 - -

8 KLHL4 kelch-like 4 (Drosophila) -1.83 -2.08 -1.96 - -

9 FBN1 fibrillin 1 -1.78 -1.98 -1.88 - -

10 PXDN peroxidasin homolog (Drosophila) -1.74 -1.99 -1.87 1 2

11 SLC16A6 solute carrier family 16, member 6 (monocarboxylic acid transporter 7) -2.01 -1.7 -1.86 1 1

12 DOCK8 dedicator of cytokinesis 8 -2.16 -1.55 -1.86 - -

13 C7orf58 chromosome 7 open reading frame 58 -2.07 -1.62 -1.84 - -

14 INSL4 insulin-like 4 (placenta) -1.53 -2.12 -1.83 - -

15 ZP3 zona pellucida glycoprotein 3 (sperm receptor) -1.76 -1.89 -1.83 - -

16 C4orf47 chromosome 4 open reading frame 47 -1.67 -1.97 -1.82 - -

17 CCDC113 coiled-coil domain containing 113 -1.75 -1.83 -1.79 - -

18 STXBP4 syntaxin binding protein 4 -1.64 -1.83 -1.74 3 2

19 PNP purine nucleoside phosphorylase -1.66 -1.67 -1.67 1 1

20 LAMP2 lysosomal-associated membrane protein 2 -1.69 -1.61 -1.65 - -

21 SGK1 serum/glucocorticoid regulated kinase 1 -1.74 -1.56 -1.65 - 1

22 BBS1 Bardet-Biedl syndrome 1 -1.68 -1.59 -1.64 - -

23 TRIM4 tripartite motif-contaning 4 -1.55 -1.6 -1.58 - 1

Down-regulated genes by miR-1 and miR-133a in IMC-3

Expression levels of miR-1 and miR-133a in clinical HNSCC samples

miR-1 expression

(Normalized to RNU48)

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0

p=0.0023

Normal Tumor

0.0050.0100.0150.0200.0250.0300.035

0

0.040p=0.0382

miR-133a expression

(Normalized to RNU48)

Normal Tumor miR-1 expression0 0.02 0.04

0

0.025

0.125

miR-133a

expression

0.050

0.075

0.100

r=0.571p<0.001

miR-1 and miR-133a expression levels in tumor tissue were significantly down-regulated in clinical HNSCC samples. Furthermore, miR-1 and miR-133a expression levels were positively correlated in HNSCC samples.

61.0M 61.1M 61.2M 61.3M

61147660 61167971

RPL7P3LOC100131174

LOC100505735FLJ32154

LOC100127888

C20orf151 GATA5 C20orf166-AS1

C20orf166 SLCO4A1

10.5KbmiR-1-1 miR-133a-2

Exon1 2 3 4

Chromosome 20q13.33

19.2M 19.3M 19.4M 19.5M

19321545 19450914

SNRPD1 ABHD3 MIB1

3.2Kb

Exon 1 21

Chromosome 18q11.2

miR-320c-1 RPL34P32

miR-133a-1 miR-1-2

2 3 154 56 7 8 9 10 11 12 13 14 1617 18 1920

Our expression signatures of human cancer including HNSCC revealed that the expression of microRNA-1 (miR-1) and microRNA-133a (miR-133a) were significantly reduced in cancer cells. In human genome, miR-1 and miR-133a located same chromosomal regions (miR-1-2 and miR-133a-1 on 18q11.2, and miR-1-1 and miR-133a-2 on 20q13.33) called cluster. Previously, our group reported that miR-1 and miR-133a function as tumor suppressors in several types of cancers including HNSCC. In this study, we identify the novel molecular networks regulated by miR-1 and miR-133a commonly in HNSCC. E

arly apoptosis cells

(relative to mock)

miR-1 and miR-133a Inhibited cell growth and inducedapoptosis in IMC-3 (derived from maxillary sinus SCC)

Normal Tumor0

1.0

2.0p=0.0089

PNP expression

(Normalized to GUSB)

0

1.0

2.0

3.0

4.0

p=0.0321

Normal TumorTAGLN2 expression

(Normalized to GUSB)

r=-0.322p=0.043

miR-10 0.02

0

PNP mRNA

0.04

1

2

miR-133a0 0.05 0.10 0.15

0

PNP mRNA

1

2r=-0.266p=0.098

miR-10 0.02

0

2

4

TAGLN2 mRNA

0.04

r=-0.407p=0.009

miR-133a0

0

2

4

TAGLN2 mRNA

0.05 0.10

r=-0.341p=0.031

0.15

Overexpression of TAGLN2 and PNP in clinical HNSCC samples

Inverse correlation between TAGLN2 and miR-1/miR-133a

Inverse correlation between PNP and miR-1/miR-133a

0

1

2

3

4

5

6

*

*

7

Cell proliferation

(% of mock)

0

20

40

60

80

100

120

*

*

Mock Control miR-1 miR-133a *:p<0.05

TAGLN2 and PNP were regulated by miR-1 and miR-133a

si-TAGLN2 and si-PNP inhibited cell growth in IMC-3

TAGLN2β-actin

TAGLN2

normalized

to β-actin

020406080

100120

TAGLN2 mRNA expression

(relative to mock)

020 *406080

100120

*

TAGLN2 3’UTR length:686miR-1 target sites

miR-133a target sites

0.2k 0.4k 0.6k

PNP mRNA expression

(relative to mock)

020

*406080

100120

*

PNP

normalized

to β-actin

020406080

100120

PNP 3’UTR length:488miR-1 target sites

miR-133a target sites

0.2k 0.4k

5' ...GCUCUUUGAGAUAAUACAUUCCG... ||||||| 3' UAUGUAUGAAGAAAUGUAAGGU

5' ...AUCUAAAUCACCAGAGACCAAAC... |||||| 3' GUCGACCAACUUCCCCUGGUUU

5' ...AUAGCCAUCAAAACUGGACCAAC... ||||||| 3' GUCGACCAACUUCCCCUGGUUU

5' ...UCUUCCUUUCCCCUGGGACCAAA... ||||||| 3' GUCGACCAACUUCCCCUGGUUU

5' ...UAUAUUUUAGCAGUGACAUUCCC... ||||||| 3' UAUGUAUGAAGAAAUGUAAGGU

5' ...CCCAUGCUUACUAAU--ACAUUCCC... |||| ||||||| 3' UAUGUAUGAAGAAAUGUAAGGU

5' ...UCUGUGUCCUCCGUUCAUUCCAU... |||||| 3' UAUGUAUGAAGAAAUGUAAGGU

Mock Control miR-1 miR-133a

PNPβ-actin

*:p<0.05

*:p<0.05

Cell proliferation

(% of mock)

0

20

*

40

60

80

100

120

Cell proliferation

(% of mock)

0

20

*

40

60

80

100

120

Mock

Control

si-TAGLN2

Mock

Control

si-PNP

*:p<0.05*:p<0.05