Download miR-375

Abstract #137
Molecular networks regulated by tumor suppressive microRNA-375 in head and neck squamous cell carcinoma
Takashi Kinoshita1,2, Toyoyuki Hanazawa1, Nijiro Nohata1,2, Naoko Kikkawa1, Miki Fuse2, Takeshi Chiyomaru3,
Hirofumi Yoshino3, Hideki Enokida3, Masayuki Nakagawa3, Yoshitaka Okamoto1, Naohiko Seki2
1Department
of Otorhinolaryngology / Head and Neck Surgery, Graduate School of Medicine, Chiba University, Chiba Japan
2Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan
3Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
miRNA
2.40E-02
7.25E-05
1.52E-04
1.20E-02
7.15E-04
6.53E-03
2.76E-03
7.15E-04
3.66E-03
1.20E-02
1
2
3
4
5
6
7
8
9
10
miR-874
miR-133a
miR-375
miR-204
miR-1
miR-139-5p
miR-145
miR-143
miR-486-3p
miR-146a
P-value
1.6
4.63E-02
3.30E-03
1.61E-02
5.50E-03
2.40E-02
4.40E-03
3.67E-02
1.90E-02
4.07E-02
4.10E-03
YWHAZ
12
13
14
15
16
17
18
19
20
DNAJB11
KRT8
HSP90B3P
EBPL
C6orf129
TTC7B
HSP90B1
C4orf3
SDF2L1
0.15
0.10
0.05
0
Normal
Tumor
Normalized to GAPDH
Normalized to GAPDH
P=0.014
0.10
+
-1.79
-1.44
-2.19
-1.8
-1.8
-2.56
-1.71
-3.04
-3.22
-2.28
-2.57
-2.53
-1.63
-2.44
-2.41
-2.33
-2.24
-2.18
-2.17
-2.1
-2.07
+
+
+
-1.23
-2.81
-2.02
+
-1.93
-2.1
-2.01
-
-1.9
-2.08
-1.99
+
-1.4
-1.78
-1.21
-1.78
-1.48
-1.44
-1.08
-1.49
-1.14
-2.22
-1.76
-2.32
-1.67
-1.95
-1.98
-2.3
-1.88
-2.15
-1.81
-1.77
-1.77
-1.73
-1.72
-1.71
-1.69
-1.69
-1.65
+
+
-
0.08
0.06
0.04
0.02
0
Normal
HERPUD1
Normal
Tumor
PSIP1
Normalized to GAPDH
P=0.12
Tumor
0.30
P=0.16
0.25
0.20
0.15
0.10
0.05
0
Normal
Tumor
Tumor
*
Position 171-177
of LDHB 3' UTR
5' ...GCAAUCUGAGCUCUU-GAACAAAU...
||||
||||||
3'
AGUGCGCUCGGCUUGCUUGUUU
Position 171-177
deletion
5' ...GCAAUCUGAGCUCUU--------U...
||||
||||||
3'
AGUGCGCUCGGCUUGCUUGUUU
1.2
1.0
0.8
0.6
0.4
0.2
0
* P<0.0001
mock
100
60
control
*
80
60
40
20
Knocking down of MTDH
inhibited cancer cell
proliferation
1454-1460
SAS
*
Position 1454-1460
of MTDH 3’UTR
5‘ ...ACUAGGAAAGCUAAACGAACAAA...
|||
|||||||
3’
AGUGCGCUCGGCUUGCUUGUUU
Position 1454-1460
deletion
5‘ ...ACUAGGAAAGCUAAA-------A...
|||
|||||||
3’
AGUGCGCUCGGCUUGCUUGUUU
0
MTDH
*
miR-375
MTDH (NM_178812) 3’UTR length:1637
40
20
100
DEL-3’UTR
miR-375 target site
80
* P<0.0001
0
SAS
* P<0.0001
120
*
WT-3’UTR
120
120
100
80
60
40
20
0
Normal
IMC-3
miR-375 directly regulates MTDH expression
both in mRNA and protein levels
0.015
0
171-177
Knocking down of LDHB
inhibited cancer cell
proliferation
* P<0.0001
120
100
80
60
40
20
0
β-Actin
0.005
miR-375 target site
GAPDH
0.020
0.010
Tumor
LDHB (NM_002300) 3’UTR length:201
LDHB
SLC7A11
P=0.38
0
1.2
1.0
0.8
0.6
0.4
0.2
0
* P<0.0001
* P<0.0001
100
80
mock
DEL-3’UTR
control
miR-375
*
40
20
0
*
WT-3’UTR
*
60
si-control
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0
P=0.015
0
0.5
mock
1. Tumor suppressive microRNA-375 regulates oncogene AEG-1
/MTDH in head and neck squamous cell carcinoma (HNSCC)
Journal of Human Genetics, 2011 56:595-601.
2. Tumor suppressive microRNA-375 regulates lactate
dehydrogenase B in maxillary sinus squamous cell carcinoma
International Journal of Oncology, 2012 40:185-93.
0.20
20
1.0
si-MTDH_2
References
-2.92
MTDH
40
1.5
si-MTDH_1
miR-375 functions as a tumor suppressor in HNSCC. LDHB and
MTDH are directly regulated by miR-375. These genes may
function as oncogenes and contributed to cell proliferation in
HNSCC. Tumor suppressive miR-375 and its target oncogenes
may provide new insights into the molecular networks of HNSCC.
Normalized to GAPDH
Conclusions
-4.12
The mRNA expression of candidate genes for miR-375 target
in 20 pairs of HNSCC samples
LDHB
0.2
Luminescence
(normalized)
11
-1.71
LDHB mRNA expression
(% of mock)
KRT5
0.4
IMC-3
160
140
120
100
80
60
40
20
0
LDHB normalized
to GAPDH
10
0.6
60
2.0
si-LDHB_2
SLC7A11
0.8
80
si-LDHB_1
9
1.0
*
si-control
6. MTDH, which promotes tumorigenesis by modulating
multiple signal transduction pathways such as NF- κB,
PI3K/AKT, and Wnt pathway, promotes cell proliferation in
HNSCC.
PDIA4
HSPA5
LDHB
NUCB2
PSIP1
CXCR7
MTDH
*
mock
5. LDHB, which is the glycolytic enzyme that catalyze the
formation of lactic acid from pyruvate, promotes cell
proliferation in HNSCC.
2
3
4
5
6
7
8
homocysteine-inducible, endoplasmic reticulum stress-inducible,
ubiquitin-like domain member 1
protein disulfide isomerase family A, member 4
heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa)
lactate dehydrogenase B
nucleobindin 2
PC4 and SFRS1 interacting protein 1
chemokine (C-X-C motif) receptor 7
metadherin
solute carrier family 7, (cationic amino acid transporter, y+
system) member 11
keratin 5
tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation
protein, zeta polypeptide
DnaJ (Hsp40) homolog, subfamily B, member 11
keratin 8
heat shock protein 90kDa beta (Grp94), member 3 (pseudogene)
emopamil binding protein-like
chromosome 6 open reading frame 129
tetratricopeptide repeat domain 7B
heat shock protein 90kDa beta (Grp94), member 1
chromosome 4 open reading frame 3
stromal cell-derived factor 2-like 1
log2 ratio
miR-375
FaDu
Average target
site
MTDH mRNA expression
(% of mock)
4. qRT-PCR, Western blots, and luciferase assays revealed that
miR-375 directly inhibits LDHB and MTDH expression.
HERPUD1
SAS
1.2
2.5
miR-375 directly regulates LDHB expression
both in mRNA and protein levels
MTDH normalized
to β-Actin
3. Genome-wide molecular targets search and TargetScan
database indicated that LDHB (lactate dehydrogenase B)
and MTDH (metadherin) were candidate genes of miR-375
target.
1
Gene Name
Normalized to GAPDH
2. Gain-of-function analysis revealed that cancer cell
proliferation was inhibited and apoptosis was induced in
miR-375 transfected HNSCC cells.
Gene
Symbol
100
Normal
Top 20 down-regulated genes by transfection of miR-375
in HNSCC cell lines
No.
* P<0.0001
1.4
0
Key Findings
1. qRT-PCR revealed that miR-375 was down-regulated in
HNSCC tissues compared with adjacent normal epithelium in
20 patients with HNSCC.
SAS
Cell proliferation
(% of mock)
miR-1
miR-375
miR-139-5p
miR-504
miR-125b
miR-199b
miR-100
miR-497
let-7c
miR-30a*
No.
P=0.008
Cell proliferation
(% of mock)
1
2
3
4
5
6
7
8
9
10
P-value
miR-375 inhibited cancer cell
proliferation and induced apoptosis
Early apoptosis cell
(relative to mock)
miRNA
Fold
Change
0.011
0.017
0.035
0.045
0.054
0.132
0.167
0.177
0.183
0.253
miR-375 expression in
20 pairs of HNSCC samples
Cell proliferation
(% of mock)
No.
Fold
Change
0.007
0.033
0.092
0.147
0.232
0.268
0.274
0.278
0.282
0.318
Top 10 down-regulated miRNAs
from TaqMan LDA in MSSCC
Luminescence
(normalized)
MicroRNAs (miRNAs) constitute a class of small non-protein
coding RNA molecules, 19 – 22 nucleotides in length. They
negatively regulate multiple genes by mRNA cleavage or
translational repression. Down-regulated miRNAs in cancer cells
could function as a tumor suppressors by negatively regulating
oncogenes. Our miRNA expression signatures of hypopharyngeal
squamous cell carcinoma (HSCC), maxillary sinus squamous cell
carcinoma (MSSCC) and esophageal squamous cell carcinoma
(ESCC) revealed that microRNA-375 (miR-375) was significantly
down-regulated in cancer tissues compared to normal epithelium.
The aim of this study was to clarify the functional significance
of miR-375 and to identify the gene targets miR-375 regulates
in head and neck squamous cell carcinoma (HNSCC).
Top 10 down-regulated miRNAs
from TaqMan LDA in HSCC
miR-375 expression
(Normalized to RNU48)
Background and Aims