Tumor suppressive microRNA‑138 contributes to cell migration and invasion through its targeting of vimentin in renal cell carcinoma.

Abstract

Many studies have recently suggested that microRNAs (miRNAs) contribute to the development of various types of human cancers as well as to their invasive and metastatic capacities. Previously, our miRNA expression signature of renal cell carcinoma (RCC) revealed that microRNA‑138 (miR‑138) was significantly reduced in cancer cells. The aim of the present study was to investigate the functional significance of miR‑138 and to identify its target genes in RCC cells. Restoration of mature miR‑138 in two RCC cell lines (A498 and 786‑O) caused changes in the bleb-like cell morphology, characteristics of the epithelial-mesenchymal transition (EMT). Restoration also significantly inhibited migration and invasion in the two RCC cell lines, suggesting that miR‑138 functions as a tumor suppressor. Genome-wide gene expression analysis (miR‑138 transfectants and RCC clinical specimens) and TargetScan database studies showed that vimentin (VIM) is a promising candidate target gene of miR‑138. It is well known that VIM is one of the most widely expressed mammalian intermediate filament proteins. Recent studies showed that VIM functions in cell adhesion, migration, survival and cell signaling processes via dynamic assembly/disassembly in cancer cells. We focused on VIM and investigated whether VIM was regulated by tumor suppressive miR‑138 and contributed to cancer cell migration and invasion in RCC cells. Restoration of miR‑138 in RCC cell lines suppressed VIM expression at both the mRNA and protein levels. Silencing studies of VIM in RCC cell lines demonstrated significant inhibition of cell migration and invasion activities in si-VIM transfectants. In clinical specimens of RCC, the expression levels of VIM were significantly upregulated in cancer tissues compared to adjacent non-cancerous tissues. Furthermore, immunohistochemistry showed that VIM expression levels in RCC specimens were significantly higher than those in normal renal tissues. These data suggest that VIM may function as an oncogene and is regulated by tumor suppressive miR‑138. The existence of a tumor suppressive miR‑138-mediated oncogenic pathway provides new insights into the potential mechanisms of RCC oncogenesis and metastasis.

Full text

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INTERNATIONAL JOURNAL OF ONCOLOGY 41: 805-817, 2012
Abstract. Many studies have recently suggested that
microRNAs (miRNAs) contribute to the development of various
types of human cancers as well as to their invasive and metastatic
capacities. Previously, our miRNA expression signature of renal
cell carcinoma (RCC) revealed that microRNA‑138 (miR‑138)
was significantly reduced in cancer cells. The aim of the present
study was to investigate the functional significance of miR‑138
and to identify its target genes in RCC cells. Restoration of
mature miR‑138 in two RCC cell lines (A498 and 786-O)
caused changes in the bleb-like cell morphology, characteristics
of the epithelial-mesenchymal transition (EMT). Restoration
also significantly inhibited migration and invasion in the two
RCC cell lines, suggesting that miR‑138 functions as a tumor
suppressor. Genome-wide gene expression analysis (miR‑138
transfectants and RCC clinical specimens) and TargetScan
database studies showed that vimentin (VIM) is a promising
candidate target gene of miR‑138. It is well known that VIM
is one of the most widely expressed mammalian intermediate
filament proteins. Recent studies showed that VIM functions in
cell adhesion, migration, survival and cell signaling processes
via dynamic assembly/disassembly in cancer cells. We focused
on VIM and investigated whether VIM was regulated by tumor
suppressive miR‑138 and contributed to cancer cell migration
and invasion in RCC cells. Restoration of miR‑138 in RCC cell
lines suppressed VIM expression at both the mRNA and protein
levels. Silencing studies of VIM in RCC cell lines demonstrated
significant inhibition of cell migration and invasion activities in
si-VIM transfectants. In clinical specimens of RCC, the expres-
sion levels of VIM were significantly upregulated in cancer tissues
compared to adjacent non-cancerous tissues. Furthermore,
immunohistochemistry showed that VIM expression levels in
RCC specimens were significantly higher than those in normal
renal tissues. These data suggest that VIM may function as an
oncogene and is regulated by tumor suppressive miR‑138. The
existence of a tumor suppressive miR‑138-mediated oncogenic
pathway provides new insights into the potential mechanisms of
RCC oncogenesis and metastasis.
Introduction
Renal cell carcinoma (RCC) is the most common neoplasm of the
adult kidney. In this disease, cancer cells form in the tubules of the
kidney and approximately 80% of RCC patients are diagnosed
with the clear cell RCC subtype (1). Up to 30% of RCC patients
present at advanced stages, and approximately 40% of patients
who undergo curative surgical resection experience recurrence
during subsequent follow-up (2,3). The five-year survival rate
of advanced RCC is 5-10% (4). RCC is resistant to radiotherapy
and chemotherapy (5,6). Targeted therapies such as sunitinib,
sorafenib, everolimus and temsirolimus have been developed
and have been used widely in first- and second-line treatments,
extending the period of progression-free-survival (7,8). However,
these treatments are insufficient for patients who have developed
relapse or metastasis. Therefore, increased understanding of the
molecular mechanisms of RCC progression and metastasis is
needed using the latest approaches to genomic analysis.
RNA can be divided into two categories: protein coding RNA
and non-coding RNA (ncRNA). It is important to examine the
functions of ncRNAs and their association with human disease,
including cancer. microRNAs (miRNAs) are endogenous small
ncRNA molecules (19-22 bases in length) that regulate protein
coding gene expression by repressing translation or cleaving
RNA transcripts in a sequence-specific manner (9). A growing
body of evidence suggests that miRNAs are aberrantly expressed
in many human cancers, and that they play significant roles in
their initiation, development, and metastasis (10). Some highly
expressed miRNAs could function as oncogenes by repressing
tumor suppressors, whereas low level miRNAs could function
as tumor suppressors by negatively regulating oncogenes (11).
Tumor suppressive microRNA‑138 contributes to cell migration
and invasion through its targeting of vimentin
in renal cell carcinoma
TAKESHI YAMASAKI1, NAOHIKO SEKI2, YASUTOSHI YAMADA1, HIROFUMI YOSHINO1,
HIDEO HIDAKA1, TAKESHI CHIYOMARU1, NIJIRO NOHATA2, TAKASHI KINOSHITA2,
MASAYUKI NAKAGAWA1 and HIDEKI ENOKIDA1
1Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima;
2Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan
Received March 28, 2012; Accepted May 30, 2012
DOI: 10.3892/ijo.2012.1543
Correspondence to: Dr Hideki Enokida, Department of Urology,
Graduate School of Medical and Dental Sciences, Kagoshima
University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
E-mail: enokida@m.kufm.kagoshima-u.ac.jp
Key words: microRNA, miR‑138, vimentin, renal cell carcinoma

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YAMASAKI et al: TUMOR SUPPRESSIVE microRNA-138 IN RENAL CELL CARCINOMA
806
We previously identified many tumor suppressive miRNAs
based on our miRNA expression signatures of various types of
cancer, such as RCC, bladder cancer, prostate cancer, maxillary
sinus squamous cell carcinoma and hypopharyngeal squamous
cell carcinoma (12-17). In oncogenic pathways, normal regula-
tory mechanisms are disrupted by aberrant expression of tumor
suppressive or oncogenic miRNAs. Therefore, identification of
miRNA-regulated pathways is important for further development
in human cancer research. Thus, we have been investigating how
tumor suppressive miRNA regulates novel cancer pathways. For
example, the miR‑1/miR‑133a cluster regulates several oncogenic
genes, including transgelin-2 (TAGLN2), prothymosin α (PTMA)
and purine nucleoside phosphorylase (PNP) (14,15,18).
More recently, we constructed a miRNA expression signa-
ture of RCC clinical specimens and successfully identified tumor
suppressive miR‑1285 targeting transglutaminase 2 (TGM2)
(12). Among the signatures, several miRNAs were significantly
downregulated in RCC specimens as promising candidate
of tumor suppressors. In this study, we focused on miR‑138.
This miRNA was downregulated in our previous signature,
and downregulation of miR‑138 has been observed in several
malignancies, including anaplastic thyroid carcinoma (19) and
lung cancer (20).
The aim of the study was to investigate the functional signif-
icance of miR‑138 and identify its target genes in RCC cells.
To identify miR‑138-regulated cancer pathways, we undertook
both a genome-wide gene expression analysis (miR‑138 trans-
fectants and RCC clinical specimens) and an in silico study.
The results showed that vimentin (VIM) was a promising
candidate target gene of miR‑138. It is well known that VIM
is one of the most widely expressed mammalian intermediate
filament proteins. Studies have shown that VIM functions in
cell adhesion, migration, survival, and cell signaling processes
via dynamic assembly/disassembly in cancer cells (21). The
existence of a tumor suppressive miR‑138-mediated cancer
pathway provides new insights into the potential mechanisms
of RCC oncogenesis and metastasis.
Materials and methods
Clinical specimens. A total of 33 pairs of clear cell renal cell
carcinoma (ccRCC) and adjacent non-cancerous specimens
were collected from patients who had undergone radical
nephrectomies at Kagoshima University Hospital. The samples
were processed and stored in RNAlater (Qiagen, Valencia, CA,
USA) at -20˚C until RNA extraction. The patient information is
summarized in Table I. These samples were staged according to
the American Joint Committee on Cancer-Union Internationale
Contre le Cancer (UICC) tumour-node-metastasis classification
and histologically graded (22). Our study was approved by the
Bioethics Committee of Kagoshima University; written prior
informed consent and approval were given by the patients.
Cell culture and RNA extraction. We used two human RCC
cell lines, A498 and 786-O, obtained from the American Type
Culture Collection (Manassas, VA, USA). The cell lines were
incubated in RPMI-1640 medium supplemented with 10% fetal
bovine serum (FBS) and maintained in a humidified incubator
(5% CO2) at 37˚C. Total-RNA was extracted, as previously
described (12).
Quantitative real‑time RT‑PCR. TaqMan probes and primers
for VIM (P/N: Hs00185584_m1: Applied Biosystems) were
assay-on-demand gene expression products. All reactions were
performed in duplicate, and a negative control lacking cDNA
was included. We followed the manufacturer's protocol for
PCR conditions. Stem-loop RT-PCR (TaqMan MicroRNA
Assays; P/N: 002284 for miR-138; Applied Biosystems) was
used to quantitate miRNAs according to the earlier published
conditions (23). To normalize the data for quantification
of VIM mRNA and the miRNAs, we used human GUSB
(P/N: Hs99999908_m1; Applied Biosystems) and RNU6B
(P/N: 001973; Applied Biosystems), respectively, and we used
the ∆∆Ct method to calculate the fold-change. As a control
RNA, we used Premium total-RNA from normal human kidney
(AM 7976; Applied Biosystems).
Mature miRNA and siRNA transfection. As described
elsewhere (23), the RCC cell lines were transfected with
Lipofectamine™ RNAiMAX transfection reagent (Invitrogen,
Carlsbad, CA, USA) and Opti-MEM™ (Invitrogen) with 10 nM
mature miRNA molecules. Pre-miR™ (Applied Biosystems)
and negative-control miRNA (Applied Biosystems) were used
in the gain-of-function experiments, whereas VIM siRNA (Cat
nos. SASI_ Hs01_00044033 and SASI_HS01_00044036,
Sigma-Aldrich, St. Louis, MO, USA) and negative control
siRNA (D-001810-10; Thermo Fisher Scientific, Waltham, MA,
USA) were used in the loss-of-function experiments. Cells were
seeded in 10-cm dishes for protein extraction (8x105 cells per
Table I. Patient characteristics of RT-PCR experiments.
No. of patients (%)
Total number
Age (average)
Gender
Male
Female
Pathological tumor stage
pT1a
pT1b
pT2
pT3a
pT3b
pT4
Grade
G1
G2
G3
Unknown
Infiltration
α
β
γ
Venous invasion
v (-)
v (+)
33
36-83 (65.6)
22
11
(66.7)
(33.3)
12
14
2
3
2
0
(36.4)
(42.4)
(6.1)
(9.1)
(6.1)
(0.0)
5
26
0
2
(15.2)
(78.8)
(0.0)
(6.1)
12
21
0
(36.4)
(63.6)
(0.0)
24
9
(72.7)
(27.3)

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INTERNATIONAL JOURNAL OF ONCOLOGY 41: 805-817, 2012
807
Table II. Downregulated genes in microRNA‑138 transfectants.
Entrez
gene ID


Target
site Symbol Average
3569
4856
84448
3773
6352
4316
3038
91543
5806
64220
84419
144406
4493
718
10964
3990
9121
4490
8091
1803
6288
4502
8638
9582
4500
3437
9076
8743
3433
2172
23586
4982
259307
6590
5174
51015
3434
22822
2537
392636
81610
26154
4940
5359
6236
4496
4814
11309
158158
259
2669
IL6
NOV
ABLIM2
KCNJ16
CCL5
MMP7
HAS3
RSAD2
PTX3
STRA6
C15orf48
WDR66
MT1E
C3
IFI44L
LIPC
SLC16A5
MT1B
HMGA2
DPP4
SAA1
MT2A
OASL
APOBEC3B
MT1L
IFIT3
CLDN1
TNFSF10
IFIT2
FABP6
DDX58
TNFRSF11B
IL4I1
SLPI
PDZK1
ISOC1
IFIT1
PHLDA1
IFI6
TMEM195
FAM83D
ABCA12
OAS3
PLSCR1
RRAD
MT1H
NINJ1
SLCO2B1
RASEF
AMBP
GEM
-5.35 interleukin 6 (interferon, β 2)
-5.22 nephroblastoma overexpressed gene
-4.97 actin binding LIM protein family, member 2
-4.86 potassium inwardly-rectifying channel, subfamily J, member 16
-4.44 chemokine (C-C motif) ligand 5
-4.1 matrix metallopeptidase 7 (matrilysin, uterine)
-4.03 hyaluronan synthase 3
-3.99 radical S-adenosyl methionine domain containing 2
-3.85 pentraxin-related gene, rapidly induced by IL-1 β
-3.85 stimulated by retinoic acid gene 6 homolog (mouse)
-3.8 chromosome 15 open reading frame 48
-3.67 WD repeat domain 66
-3.66 metallothionein 1E
-3.65 complement component 3
-3.64 interferon-induced protein 44-like
-3.64 lipase, hepatic
-3.61 solute carrier family 16, member 5 (monocarboxylic acid transporter 6)
-3.6 metallothionein 1B
-3.56 high mobility group AT-hook 2
-3.49 dipeptidyl-peptidase 4
-3.48 serum amyloid A1
-3.44 metallothionein 2A
-3.43 2'-5'-oligoadenylate synthetase-like
-3.39 apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B
-3.32 metallothionein 1L (gene/pseudogene)
-3.3 interferon-induced protein with tetratricopeptide repeats 3
-3.05 claudin 1
-3.03 tumor necrosis factor (ligand) superfamily, member 10
-2.94 interferon-induced protein with tetratricopeptide repeats 2
-2.91 fatty acid binding protein 6, ileal
-2.89 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58
-2.89 tumor necrosis factor receptor superfamily, member 11b
-2.88 interleukin 4 induced 1
-2.88 secretory leukocyte peptidase inhibitor
-2.88 PDZ domain containing 1
-2.86 isochorismatase domain containing 1
-2.84 interferon-induced protein with tetratricopeptide repeats 1
-2.79 pleckstrin homology-like domain, family A, member 1
-2.76 interferon, α-inducible protein 6
-2.76 transmembrane protein 195
-2.73 family with sequence similarity 83, member D
-2.66 ATP-binding cassette, sub-family A (ABC1), member 12
-2.65 2'-5'-oligoadenylate synthetase 3, 100 kDa
-2.65 phospholipid scramblase 1
-2.61 Ras-related associated with diabetes
-2.56 metallothionein 1H
-2.54 ninjurin 1
-2.5 solute carrier organic anion transporter family, member 2B1
-2.47 RAS and EF-hand domain containing
-2.47 α-1-microglobulin/bikunin precursor
-2.47 GTP binding protein overexpressed in skeletal muscle
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(-)
(-)
(+)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(-)
(-)
(-)
(-)
(+)
(-)
(+)
(-)
(-)
(-)
(+)
(+)
(-)
(-)
(-)

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YAMASAKI et al: TUMOR SUPPRESSIVE microRNA-138 IN RENAL CELL CARCINOMA
808
Table II. Continued.
Entrez
gene ID


Target
site Symbol Average
3656
3880
1978
7431
57568
7913
123
4501
654346
4489
3669
2920
2274
157506
25937
3690
196513
9518
1364
23643
10561
84141
6281
7088
81553
4599

6850
7364
5366
50515
2982
6273
54478
3428
9615
7849
10550
23286
9636
896
5329
4853
55652
23476
2012
3429
79710
80820
IRAK2
KRT19
EIF4EBP1
VIM
SIPA1L2
DEK
PLIN2
MT1X
LGALS9C
MT1A
ISG20
CXCL2
FHL2
RDH10
WWTR1
ITGB3
DCP1B
GDF15
CLDN4
LY96
IFI44
FAM176A
S100A10
TLE1
FAM49A
MX1

SYK
UGT2B7
PMAIP1
CHST11
GUCY1A3
S100A2
FAM64A
IFI16
GDA
PAX8
ARL6IP5
WWC1
ISG15
CCND3
PLAUR
NOTCH2
SLC48A1
BRD4
EMP1
IFI27
MORC4
EEPD1
-2.42 interleukin-1 receptor-associated kinase 2
-2.41 keratin 19
-2.41 eukaryotic translation initiation factor 4E binding protein 1
-2.39 vimentin
-2.38 signal-induced proliferation-associated 1 like 2
-2.35 DEK oncogene
-2.34 perilipin 2
-2.33 metallothionein 1X
-2.31 lectin, galactoside-binding, soluble, 9C
-2.3 metallothionein 1A
-2.28 interferon stimulated exonuclease gene 20 kDa
-2.28 chemokine (C-X-C motif) ligand 2
-2.27 four and a half LIM domains 2
-2.27 retinol dehydrogenase 10 (all-trans)
-2.26 WW domain containing transcription regulator 1
-2.26 integrin, β 3 (platelet glycoprotein IIIa, antigen CD61)
-2.24 DCP1 decapping enzyme homolog B (S. cerevisiae)
-2.24 growth differentiation factor 15
-2.23 claudin 4
-2.2 lymphocyte antigen 96
-2.2 interferon-induced protein 44
-2.19 family with sequence similarity 176, member A
-2.17 S100 calcium binding protein A10
-2.17 transducin-like enhancer of split 1 (E(sp1) homolog, Drosophila)
-2.17 family with sequence similarity 49, member A
-2.17 myxovirus (influenza virus) resistance 1,
interferon-inducible protein p78 (mouse)
-2.17 spleen tyrosine kinase
-2.17 UDP glucuronosyltransferase 2 family, polypeptide B7
-2.17 phorbol-12-myristate-13-acetate-induced protein 1
-2.16 carbohydrate (chondroitin 4) sulfotransferase 11
-2.16 guanylate cyclase 1, soluble, α 3
-2.15 S100 calcium binding protein A2
-2.15 family with sequence similarity 64, member A
-2.14 interferon, γ-inducible protein 16
-2.14 guanine deaminase
-2.13 paired box 8
-2.11 ADP-ribosylation-like factor 6 interacting protein 5
-2.1 WW and C2 domain containing 1
-2.08 ISG15 ubiquitin-like modifier
-2.07 cyclin D3
-2.07 plasminogen activator, urokinase receptor
-2.06 Notch homolog 2 (Drosophila)
-2.05 solute carrier family 48 (heme transporter), member 1
-2.04 bromodomain containing 4
-2.03 epithelial membrane protein 1
-2.02 interferon, α-inducible protein 27
-2.01 MORC family CW-type zinc finger 4
-2.01 endonuclease/exonuclease/phosphatase family domain containing 1
(-)
(-)
(+)
(+)
(-)
(+)
(-)
(-)
(+)
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(+)
(+)
(-)
(-)
(-)
(-)
(+)
(+)
(+)
(+)
(-)
(+)
(+)
(+)
(+)
(-)
(-)
(+)

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dish), 6-well plates for wound healing assays (20x104 cells per
well), in 24-well plates for the mRNA extraction and Matrigel
invasion assays (5x104 cells per well) and in 96-well plates for
the XTT assays (3,000 cells per well).
Cell morphology. Cells were transfected with miR‑138 and
si-VIM for 72 h and were then examined by an inverted micro-
scope (CK2-BIP2, Olympus).
Cell proliferation, migration and invasion assays. Cell prolif-
eration was determined using an XTT assay (Roche Applied
Science, Tokyo, Japan) that was performed according to
the manufacturer's instructions. Cell migration activity was
evaluated with a wound healing assay. Cells were plated in
6-well dishes and the cell monolayer was scraped using a P-20
micropipette tip. The initial gap length (0 h) and the residual
gap length 24 h after wounding were calculated from photomi-
crographs. A cell invasion assay was carried out using modified
Boyden Chambers consisting of Transwell-precoated Matrigel
membrane filter inserts with 8-mm pores in 24-well tissue
cultures plates (BD Bioscience, Bedford, MA, USA). Minimum
essential medium containing 10% FBS in the lower chamber
served as the chemoattractant as described previously (24). All
experiments were performed in triplicate.
Screening of miR‑138‑regulated genes by microarray. Oligo-
microarray Human 60K (Agilent) was used for expression
signature in miR‑138-transfected A498 cells in comparison
with the miR-negative control transfectant, as previously
described (23). Briefly, hybridization and washing steps were
performed in accordance with the manufacturer's instructions.
The arrays were scanned using a Packard GSI Lumonics
ScanArray 4000 (PerkinElmer, Boston, MA, USA). The data
obtained were analyzed with DNASIS array software (Hitachi
Software Engineering, Tokyo, Japan) that converted the signal
intensity. Data from each microarray study were normalized by
global normalization.
Expression signature of RCC clinical specimens by microarray.
Oligo-microarray Human 60K (Agilent) was used for expres-
sion signature in 5 pairs of RCC clinical specimens compared
with adjacent non-cancerous tissues. Their age ranged from 42
to 77 years; 3 were G1 and 2 were G2 in their tumor grading;
and all were pT1N0M0 tumors.
Western blot analysis. After three days of transfection, protein
lysates (40 µg) were separated by NuPAGE on 4-12% bis-tris
gels (Invitrogen) and transferred onto polyvinylidene fluoride
membranes. Immunoblotting was done with diluted (1:500)
polyclonal VIM antibody (HPA001762; Sigma-Aldrich) and
GAPDH antibody (MAB374; Chemicon, Temecula, CA, USA).
The membrane was washed and then incubated with goat anti-
rabbit IgG (H+L)-HRP conjugate (Bio-Rad, Hercules, CA, USA).
Specific complexes were visualized with an echochemilumines-
cence (ECL) detection system (GE Healthcare, Little Chalfont,
UK), and the expression levels of these genes were evaluated by
ImageJ software (ver. 1.43; http://rsbweb.nih.gov/ij/index.html).
Immunohistochemistry. A tissue microarray of 67 RCC samples
and 10 normal kidney samples was obtained from US Biomax
Inc. (KD806; Rockville, MD, USA). Detailed information on
all tumor specimens can be found at http://www.biomax.us/
index.php. Patient characteristics are summarized in Table III.
Immunostaining was done on the tissue microarray following
the manufacturer's protocol by UltraVision Detection System
(Thermo Scientific). The primary rabbit polyclonal antibodies
against VIM (Sigma-Aldrich) were diluted 1:500. The slides were
treated with biotinylated goat anti-rabbit. Diaminobenzidine
hydrogen peroxidase was the chromogen, and the counterstaining
was done with 0.5% hematoxylin. Immunostaining was evalu-
ated according to a scoring method described previously (14).
Each case was scored on the basis of the intensity and area of
staining. The intensity of staining was graded on the following
scale: 0, no staining; 1+, mild staining; 2+, 30-60% stained
positive; 3+, >60% stained positive. A combined staining score
(intensity + extent) of <2 was low expression, a score between 3
and 4 was moderate expression, and a score between 5 and 6 was
high expression.
Statistical analysis. The relationships between two variables
and numerical values were analyzed using the Mann-Whitney
U test, and the relationship between three variables and the
numerical values was analyzed using the Bonferroni-adjusted
Mann-Whitney U test. Expert Stat View analysis software
(ver. 4; SAS institute Inc., Cary, NC, USA) was used in both
analyses. In the comparison of three variables, a non-adjusted
statistical level of significance of P<0.05 corresponded to the
Bonferroni-adjusted level of P<0.0167.
Results
Effect of miR‑138 transfection on cell proliferation, migration,
and invasion activity of RCC cell lines. In this study, we firstly
observed that restoration of miR‑138 in RCC cell lines (A498
and 786-O) changed the bleb-like cell morphology characteristic
of the epithelial-mesenchymal transition (EMT) (Fig. 1A). A
morphological change of cancer cells by miRNA transfection is
an important discovery and it suggested that miR‑138 functions
Table III. Patient characteristics of immunohistochemistry.
No. of patients (%)
Total number
Age (average)
Gender
Male
Female
Pathological tumor stage
pT1
pT2
pT3
pT4
Grade
G1
G2
G3
67
30-80 (54.4)
45
22
(67.2)
(32.8)
15
28
22
2
(22.4)
(41.8)
(32.8)
(3.0)
52
14
1
(77.6)
(20.9)
(1.5)
Normal tissue 10

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Figure 1. Effect of miR‑138 transfection on RCC cell lines. (A) The change of morphology of miR‑138 transfectants. A498 and 786-O cells were transfected
with miR‑138 for 72 h and were then examined by an inverted microscope. (B) miR‑138 expression in A498 and 786-O cell lines and in normal kidney. miR‑138
expression levels in A498 and 786-O were significantly lower than those in normal human kidney RNA. RNU6B was used as an internal control. (C-E) Effect of
miR‑138 transfection of A498 and 786-O cells. (C) Cell proliferation determined by the XTT assay; (D) cell migration activity determined by wound healing assay;
and (E) cell invasion activity determined by the Matrigel invasion assay. *P<0.001, **P<0.0001.

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Figure 2. miR‑138 regulates molecular targets in RCC cells. (A) Heatmap derived from five RCC samples. A total of 99 genes were downregulated less than -2.0-fold
in miR‑138 transfectants. We checked their mRNA expression levels in RCC by using our previous gene expression analysis of five RCC specimens. Ninety-nine
gene expression levels are shown in the heat map diagram. VIM was the top upregulated gene among the genes which have miR-138 target sites in the heat map
diagram. (B) VIM mRNA expression after 24 h transfection with 10 nM miR‑138. (C) VIM protein expression after 72 h transfection of miRNAs. GAPDH was
used as a loading control. The mRNA and protein levels of VIM were repressed in the transfectants. *P<0.01, **P<0.0001, (D) miR-138 binding sites in the 3'UTR
of VIM mRNA.

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as a tumor suppressor in RCC cells. To explore that possibility,
the following experiments were conducted.
We evaluated the expression levels of miR‑138 in two RCC
cell lines, A498 and 786-O. RNA was extracted and miRNA
expression levels of miR‑138 were determined by real-time
RT-PCR. The expression levels of miR‑138 were significantly
lower in both RCC cell lines compared with normal kidney RNA
(relative to normal kidney RNA, 0.090±0.008 and 0.102±0.009,
respectively) (Fig. 1B).
The XTT assay revealed that cell proliferation was signifi-
cantly inhibited in miR‑138 transfectants in comparison with
the transfectant reagent only (mock) and the miR-control
transfectants. The percentages of cell proliferation for A498
were 94.6±0.9, 100.0±0.8 and 100.0±1.0, respectively, each
P=0.0008. For 786-O, the percentages were 83.5±1.1, 100.0±0.4
and 100.3±0.6, respectively, P<0.0001 (Fig. 1C).
The wound healing assay demonstrated that significant inhi-
bition of cell migration occurred in the miR‑138 transfectants in
comparison with mock and the miR-control transfectants. The
percentages of wound closure for A498 were 6.5±2.3, 100.0±2.7
and 104.8±4.9, respectively, each P<0.0001. For 786-O, the
percentages were 30.7±3.8, 100.0±4.4 and 95.9±5.4, respectively,
each P<0.0001 (Fig. 1D).
The Matrigel invasion assay demonstrated that the number
of invading cells significantly decreased in the miR‑138-trans-
fectants in comparison with mock and the miR-control
transfectants. The percentages of cell invasion for A498 were
0.9±0.4, 100.0±8.6 and 83.1±7.4, respectively, each P<0.0001.
For 786-O, the values were 10.9±1.1, 100.0±4.4 and 75.3±6.2,
respectively, each P<0.0001 (Fig. 1E).
miR‑138 regulation of molecular targets assessed by genome‑
wide gene expression analysis. To confirm that miR‑138
regulated molecular targets in RCC cells, we performed
genome-wide gene expression analysis using miR‑138 trans-
fectants compared with miRNA-control transfectants in A498
cells. A total of 99 genes were downregulated in miR‑138 trans-
fectants. Among them, 24 genes had putative target site(s) in
their 3' untranslated region (3'UTR) according to the TargetScan
miRNA program (Table II).
Furthermore, we performed gene expression analysis
using RCC clinical specimens (5 pairs of RCC and adjacent
non-cancerous tissues). Several protein-coding genes were
differentially expressed in this signature (data not shown). We
selected 99 genes that were downregulated in miR‑138 trans-
fectants and demonstrated their expression levels in a heatmap
diagram (Fig. 2A). Entries from the microarray data were
approved by the Gene Expression Omnibus (GEO), and were
assigned GEO accession numbers GSE 36951 (RCC clinical
specimens) and GSE 37119 (miR‑138 transfectants).
The two expression signatures in this study (miR‑138
transfectants and RCC clinical specimens) revealed that VIM
was a promising putative target gene in miR‑138 in RCC. Thus,
we focused on the VIM gene and investigated the functional
significance of VIM in RCC cells.
VIM as a direct target of repression by miR‑138 in RCC cells.
The mRNA and protein expression levels of VIM were mark-
edly downregulated in miR‑138 transfectants (A498 and 768-O)
in comparison with the mock and miRNA-control transfectants
(Fig. 2B and C). The predicted target site of miR‑138 in VIM in
the 3'UTR is shown in Fig. 2D.
Silencing of VIM in RCC cell lines and the effect on cell
proliferation, migration and invasion. First, we assessed the
expression level of VIM in cells to be used for functional analysis
of VIM. VIM mRNA expression levels in A498 and 786-O were
significantly higher than those in normal human kidney RNA
(relative to normal kidney RNA, 4.879±0.131 and 9.298±0.255,
respectively, each P<0.0001) (Fig. 3A).
Figure 3. VIM expression was suppressed by si-VIM transfection on RCC cell lines. (A) The expression of VIM mRNAs in A 498 and 786-0 cell lines and normal
kidney. The mRNA expression levels of VIM were 4- and 9-fold higher in RCC cell lines compared to the normal kidney RNA. GUSB was used as an internal
control. (B) VIM mRNA expression after 24 h of transfection with 10 nM si-VIM. VIM mRNA expression was repressed in si-VIM transfectants. GUSB was used
as an internal control. (C) VIM protein expression after 72 h transfection of si-VIM. GAPDH was used as a loading control. The expression level of VIM was also
repressed in the transfectants.

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Figure 4. Response to VIM silencing by si-VIM transfection of RCC cell lines. (A) The change of morphology of si-VIM transfectants. A498 and 786-O cells were
transfected with si-VIM for 72 h and were then examined by an inverted microscope. (B-D) VIM-knockdown effects on A498 and 786-0 cell lines transfected with
si-VIM‑1 and si-VIM‑2. (B) Cell proliferation determined by the XTT assay; (C) cell migration activity determined by the wound healing assay; and (D) cell invasion
activity determined by the Matrigel invasion assay. *P<0.005, **P<0.0001.

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To examine the functional role of VIM, we performed
loss-of-function studies using two different siRNAs, si-VIM‑1
and si-VIM‑2 transfected into A498 and 768-O cell lines. The
mRNA and protein expression levels of VIM were markedly
downregulated in both si-VIM‑1 and si-VIM‑2 transfectants
(A498 and 768-O) in comparison with the siRNA-control trans-
fectants (Fig. 3B and C). This result shows that two siRNA were
useful for loss-of-function assays in this study.
Transfection of si-VIM‑1 and si-VIM‑2 in the RCC cell
lines (A498 and 768-O) caused EMT-like changes in cell
morphology, as that observed when cells were transfected with
miR‑138 (Fig. 4A).
The XTT assay revealed that cell proliferation was inhibited
in both si-VIM-transfectants in comparison with the si-control
transfectants. The percentages of cell proliferation for A498
were 76.1±2.6, 87.5±1.9 and 100.0±3.1, respectively, P<0.0001
and P =0.0038. For 786-O, the values were 43.1±1.0, 89.9±0.7
and 100.0±1.1, respectively, P<0.0001 (Fig. 4B).
The wound healing assay demonstrated that significant
inhibition of cell migration occurred in the si-VIM-transfectants
in comparison with the si-control transfectants. The percent-
ages of wound closure for A498 were 26.0±10.6, 44.7±8.4 and
100.0±2.6, respectively, each P<0.0001. For 786-O, the values
were 60.8±7.3, 73.0±1.7 and 100.0±7.1, respectively, P<0.0001
and P=0.0002 (Fig. 4C).
The Matrigel invasion assay demonstrated that the number
of invading cells significantly decreased in the si-VIM-transfec-
tants in comparison with the si-control transfectants. For A498,
the percentages of cells invading were 26.5±4.7, 54.8±8.3 and
100.0±12.4, respectively, P<0.0001 and P=0.0019. For 786-O,
the values were 43.6±8.3, 61.6±3.3 and 100.0±11.1, respectively,
P<0.0001 and P=0.0034 (Fig. 4D).
Expression levels of miR‑138 and VIM mRNA in RCC clinical
specimens. Quantitative stem-loop RT-PCR demonstrated that
the expression levels of miR‑138 were significantly reduced in
33 RCC samples (Table I) in comparison with adjacent non-
cancerous specimens (clinical RCC specimens, 0.346±0.201
versus adjacent normal tissues, 2.983±0.715, P<0.0001) (Fig. 5A).
On the other hand, the mRNA expression level of VIM was
significantly higher in RCC than adjacent non-cancerous speci-
mens (clinical RCC specimens, 6.017±0.622, adjacent normal
tissues; 1.316±0.224, P<0.0001) (Fig. 5B).
The mRNA expression of ≥T2 specimens (n=7) was signifi-
cantly higher than that of T1 (n=26) (T1, 5.230±0.607; ≥T2,
8.773±1.349, P=0.0277) (Fig. 5C).
Immunohistochemistry of VIM in tissue microarray. VIM was
detected by immunohistochemical staining. Fig. 6A-D shows
representative results of immunohistochemical staining of
VIM. VIM was strongly expressed in tumor lesions (Fig. 6A-C),
whereas no expression was observed in normal tissue (Fig. 6D).
The expression score of VIM was significantly higher in 67 RCC
specimens in comparison with ten normal kidney specimens
(Fig. 6E). The VIM expression of ≥T2 specimens (n=52) was
higher than that of T1 (n=15). There was a trend but no significant
difference in the expression score of VIM between T1 and ≥T2
specimens (P=0.053, Fig. 6F). While there was a trend between
VIM expression and grade, the difference was not significant.
Patient characteristics are summarized in Table III.
Discussion
The incidence of RCC has increased over the last few decades,
and it currently represents approximately 2% of all cancer-related
deaths (25). Although two-thirds of RCC patients have clinically
localized disease and will undergo curative surgery, up to 40%
of patients develop distant metastasis and their outcomes are
poor (2-4). Many studies have indicated that cell adhesion and
extra-cellular matrix proteins contribute to the cells' acquired
abilities for invasion, migration and metastasis (26).
EMT is an embryologically conserved genetic program
that is an essential step enabling cancer cell invasion and
metastasis. For example, epithelial cells lose intercellular
Figure 5. The expression levels of miR‑138 and VIM in RCC clinical specimens. (A and B) miR‑138 and VIM mRNA expression levels of 33 RCC and adjacent
non-cancerous kidney tissues. Relative expression levels are expressed in box plots. (A) Expression levels of miR‑138 in RCC clinical samples were significantly
downregulated compared with adjacent normal kidney. (B) Expression levels of VIM mRNA in RCC clinical samples were significantly upregulated compared with
adjacent normal kidney samples. (C) The correlation of VIM mRNA between T1 and ≥T2 in RCC samples. VIM expression in ≥T2 RCC samples was significantly
higher compared with T1 RCC samples.

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tight junctions and polarity (27). Recent data indicate that the
microRNA‑200 family (miR‑200a, ‑200b, ‑200c, ‑141 and ‑429)
is downregulated in aggressive human cancers. Moreover, it
plays critical roles in the inhibition of key regulators of EMT
and β-catenin/Wnt signaling (28,29). Interestingly, our miRNA
expression signature of RCC showed that all miR‑200 family
members were reduced in clinical specimens and that restora-
tion of the miR‑200 family inhibited cancer cell migration and
invasion (data not shown).
Our previous study showed that miR‑138 was reduced in
RCC miRNA expression signature (12), we validated the down-
regulation of miR‑138 in RCC clinical specimens in this study.
Aberrant expression of miR‑138 has been observed in several
types of cancer such as head and neck squamous cell carci-
noma (HNSCC) (30), anaplastic thyroid carcinoma (19) and
lung cancer (20). Two miR‑138 precursor genes, miR‑138‑1 and
miR‑138‑2, have identical sequences in the mature miRNA and
map to human chromosomes 3p21.33 and 16q13, respectively.
Although it is believed that genomic deletion or epigenetic
silencing of miRNA in cancer cells, the molecular mechanism
of downregulated miRNAs in RCC is not clear. In the human
chromosomal region 3p, LOH is frequently observed in many
cancers including RCC (31,32). This problem can be solved by
genome-based high throughput analysis in each clinical case.
Figure 6. Immunohistochemical staining of VIM in tissue microarray. (A-C) Positively stained tumor lesions (A) T1 N0 M0; (B) T2 N0 M0 and (C) T3 N0 M0.
(D) Negative staining in normal kidney tissue. (E-F) VIM expression levels in immunohistochemical staining; (E) VIM expression in normal kidney and RCC;
(F) correlation between VIM expression and clinic pathologic parameters in RCC.

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Importantly, we found significant morphologic change in
RCC cell lines (A498 and 786-O) by miR‑138 transfection.
Furthermore, restoration of miR‑138 significantly inhibited
cancer cell migration and invasion in RCC cells. These data
suggested that miR‑138 functions as a tumor suppressor that
inhibits RCC invasion and metastasis. miRNAs are unique
in their ability to regulate many protein-coding genes. A
single miRNA is capable of targeting a number of genes to
regulate biological processes globally. Bioinformatic predic-
tions suggest that miRNAs regulate more than 30% of protein
coding genes (9). The elucidation of new molecular pathways
regulated by tumor suppressive miR‑138 is important for our
understanding of human RCC invasion and metastasis. Based
on this view, we performed molecular target searches for
miR‑138 in cancer cells by combining two genome-wide gene
expression studies (miR‑138 transfectants and RCC mRNA
clinical signature) and in silico analysis.
In this study, we focused on VIM as a putative candidate
of miR‑138 in RCC cells. We chose VIM for the following
reasons. First, downregulation of VIM was recognized in
the expression signature of miR‑138 transfectants. Second,
overexpression of VIM was observed in RCC clinical speci-
mens. Third, VIM has a putative miR‑138 target site in its 3'
untranslated region. Our data demonstrated that restoration
of miR‑138 significantly inhibited both mRNA and protein
expression levels of VIM in RCC cells, suggesting VIM was
regulated by tumor suppressive miR‑138. It is well known that
VIM is an essential constituent of cytoskeletal proteins of
mesenchymal cells and VIM is a marker of EMT (21). During
EMT, cytoskeletal proteins are changed from keratin-rich
networks to VIM-rich networks connected to focal adhe-
sions. Morphological changes of RCC cells and accelerated
cell migration and invasion is caused by the reduction of
miR‑138 and the upregulation of VIM pathways. Interestingly,
it has been shown that miR‑138 regulated cell migration
and invasion by targeting RhoC, ROCK, ZEB2, EZH2 and
VIM in HNSCC cells (33,34). Importantly, restoration of
miR‑138 in an HNSCC cell line changed the EMT-like cell
morphology and suppressed cell migration and invasion (34).
This result is in accord with the data obtained in our study of
RCC. Furthermore, overexpression of miR‑138 reduced cell
viability and colony formation in HCC cell lines targeting
CCND3 (35). The report also showed that protein expression
of CCND3 was negatively correlated with miR‑138 expres-
sion in HCC tissues. Our data of miR‑138 transfectants in
RCC cell lines demonstrated that CCND3 is a putative target
of miR‑138 in RCC, suggesting that miR‑138 regulation of the
CCND3 pathway is important for RCC oncogenesis.
The results of this study and previous data indicate that
VIM is a functional target of tumor suppressive miR‑138, and
this pathway contributes to cancer cell migration, invasion,
and metastasis. In this study, we also demonstrated overex-
pression of VIM in clinical specimens of RCC. Previous
studies indicated that VIM was a sensitive and specific marker
for conventional RCCs (36,37). The combination of VIM and
CD9 staining was found to distinguish clear cell RCC and
chromophobe RCC (37). Our tissue microarray data showed
a positive correlation between VIM expression and tumor
grade in RCC specimens. In this analysis, we were not able
to obtain a correlation of VIM expression and metastasis in
RCC patients. Silencing of VIM in RCC cell lines changed
cell morphology and significantly inhibited cell migration and
invasion in this study. Thus, we propose that overexpression
of VIM participates in metastasis of RCC. Studies of a large
number of samples with balanced pathological backgrounds
are needed to elucidate the precise correlation between VIM
and/or miR‑138 expression and clinicopathological parameters.
In summary, the reduction of miR‑138 and the increased
expression of VIM are frequent events in RCC clinical
specimens. Restoration of miR‑138 in RCC cells changed the
EMT-like morphology and suppressed cell migration and inva-
sion. The tumor suppressive miR‑138-mediated cancer pathway
provides new insights into the potential mechanisms of RCC
oncogenesis and metastasis.
Acknowledgements
This research was supported by the Ministry of Education,
Science, Sports and Culture Grant-in-Aid for Scientific
Research (C), 20591861 and 21592187.
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