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

Article (PDF Available)inInternational Journal of Oncology 41(3):805-17 · July 2012with60 Reads
DOI: 10.3892/ijo.2012.1543 · Source: PubMed
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.
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 (miR138)
was signicantly reduced in cancer cells. The aim of the present
study was to investigate the functional signicance of miR138
and to identify its target genes in RCC cells. Restoration of
mature miR138 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 signicantly inhibited migration and invasion in the two
RCC cell lines, suggesting that miR138 functions as a tumor
suppressor. Genome-wide gene expression analysis (miR138
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
lament 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 miR138 in RCC cell
lines suppressed VIM expression at both the mRNA and protein
levels. Silencing studies of VIM in RCC cell lines demonstrated
signicant inhibition of cell migration and invasion activities in
si-VIM transfectants. In clinical specimens of RCC, the expres-
sion levels of VIM were signicantly upregulated in cancer tissues
compared to adjacent non-cancerous tissues. Furthermore,
immunohistochemistry showed that VIM expression levels in
RCC specimens were signicantly 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 ve-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 rst- and second-line treatments,
extending the period of progression-free-survival (7,8). However,
these treatments are insufcient 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-specic manner (9). A growing
body of evidence suggests that miRNAs are aberrantly expressed
in many human cancers, and that they play signicant 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 YAMASAKI
1
, NAOHIKO SEKI
2
, YASUTOSHI YAMADA
1
, HIROFUMI YOSHINO
1
,
HIDEO HIDAKA
1
, TAKESHI CHIYOMARU
1
, NIJIRO NOHATA
2
, TAKASHI KINOSHITA
2
,
MASAYUKI NAKAGAWA
1
and HIDEKI ENOKIDA
1
1
Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima;
2
Department 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, miR138, vimentin, renal cell carcinoma
YAMASAKI et al: TUMOR SUPPRESSIVE microRNA-138 IN RENAL CELL CARCINOMA
806
We previously identied 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, identication 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/miR133a 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 identied tumor
suppressive miR1285 targeting transglutaminase 2 (TGM2)
(12). Among the signatures, several miRNAs were signicantly
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 miR138 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 miR138 and identify its target genes in RCC cells.
To identify miR138-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 miR138. It is well known that VIM
is one of the most widely expressed mammalian intermediate
lament 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 miR138-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 -2C 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 classication
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 humidied incubator
(5% CO
2
) 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 Scientic, Waltham, MA,
USA) were used in the loss-of-function experiments. Cells were
seeded in 10-cm dishes for protein extraction (8x10
5
cells per
Table I. Patient characteristics of RT-PCR experiments.
No. of patients (%)
Total number 33
Age (average) 36-83 (65.6)
Gender
Male 22 (66.7)
Female 11 (33.3)
Pathological tumor stage
pT1a 12 (36.4)
pT1b 14 (42.4)
pT2 2 (6.1)
pT3a 3 (9.1)
pT3b 2 (6.1)
pT4 0 (0.0)
Grade
G1 5 (15.2)
G2 26 (78.8)
G3 0 (0.0)
Unknown 2 (6.1)
Inltration
α 12 (36.4)
β 21 (63.6)
γ 0 (0.0)
Venous invasion
v (-) 24 (72.7)
v (+) 9 (27.3)
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 805-817, 2012
807
Table II. Downregulated genes in microRNA‑138 transfectants.
Entrez Target
gene ID Symbol Average site
3569 IL6 -5.35 interleukin 6 (interferon, β 2) (-)
4856 NOV -5.22 nephroblastoma overexpressed gene (-)
84448 ABLIM2 -4.97 actin binding LIM protein family, member 2 (-)
3773 KCNJ16 -4.86 potassium inwardly-rectifying channel, subfamily J, member 16 (-)
6352 CCL5 -4.44 chemokine (C-C motif) ligand 5 (-)
4316 MMP7 -4.1 matrix metallopeptidase 7 (matrilysin, uterine) (-)
3038 HAS3 -4.03 hyaluronan synthase 3 (+)
91543 RSAD2 -3.99 radical S-adenosyl methionine domain containing 2 (-)
5806 PTX3 -3.85 pentraxin-related gene, rapidly induced by IL-1 β (-)
64220 STRA6 -3.85 stimulated by retinoic acid gene 6 homolog (mouse) (+)
84419 C15orf48 -3.8 chromosome 15 open reading frame 48 (-)
144406 WDR66 -3.67 WD repeat domain 66 (-)
4493 MT1E -3.66 metallothionein 1E (-)
718 C3 -3.65 complement component 3 (-)
10964 IFI44L -3.64 interferon-induced protein 44-like (-)
3990 LIPC -3.64 lipase, hepatic (-)
9121 SLC16A5 -3.61 solute carrier family 16, member 5 (monocarboxylic acid transporter 6) (-)
4490 MT1B -3.6 metallothionein 1B (-)
8091 HMGA2 -3.56 high mobility group AT-hook 2 (-)
1803 DPP4 -3.49 dipeptidyl-peptidase 4 (-)
6288 SAA1 -3.48 serum amyloid A1 (-)
4502 MT2A -3.44 metallothionein 2A (-)
8638 OASL -3.43 2'-5'-oligoadenylate synthetase-like (-)
9582 APOBEC3B -3.39 apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B (-)
4500 MT1L -3.32 metallothionein 1L (gene/pseudogene) (-)
3437 IFIT3 -3.3 interferon-induced protein with tetratricopeptide repeats 3 (-)
9076 CLDN1 -3.05 claudin 1 (-)
8743 TNFSF10 -3.03 tumor necrosis factor (ligand) superfamily, member 10 (-)
3433 IFIT2 -2.94 interferon-induced protein with tetratricopeptide repeats 2 (-)
2172 FABP6 -2.91 fatty acid binding protein 6, ileal (-)
23586 DDX58 -2.89 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (-)
4982 TNFRSF11B -2.89 tumor necrosis factor receptor superfamily, member 11b (-)
259307 IL4I1 -2.88 interleukin 4 induced 1 (-)
6590 SLPI -2.88 secretory leukocyte peptidase inhibitor (-)
5174 PDZK1 -2.88 PDZ domain containing 1 (-)
51015 ISOC1 -2.86 isochorismatase domain containing 1 (+)
3434 IFIT1 -2.84 interferon-induced protein with tetratricopeptide repeats 1 (-)
22822 PHLDA1 -2.79 pleckstrin homology-like domain, family A, member 1 (-)
2537 IFI6 -2.76 interferon, α-inducible protein 6 (-)
392636 TMEM195 -2.76 transmembrane protein 195 (-)
81610 FAM83D -2.73 family with sequence similarity 83, member D (+)
26154 ABCA12 -2.66 ATP-binding cassette, sub-family A (ABC1), member 12 (-)
4940 OAS3 -2.65 2'-5'-oligoadenylate synthetase 3, 100 kDa (+)
5359 PLSCR1 -2.65 phospholipid scramblase 1 (-)
6236 RRAD -2.61 Ras-related associated with diabetes (-)
4496 MT1H -2.56 metallothionein 1H (-)
4814 NINJ1 -2.54 ninjurin 1 (+)
11309 SLCO2B1 -2.5 solute carrier organic anion transporter family, member 2B1 (+)
158158 RASEF -2.47 RAS and EF-hand domain containing (-)
259 AMBP -2.47 α-1-microglobulin/bikunin precursor (-)
2669 GEM -2.47 GTP binding protein overexpressed in skeletal muscle (-)
YAMASAKI et al: TUMOR SUPPRESSIVE microRNA-138 IN RENAL CELL CARCINOMA
808
Table II. Continued.
Entrez Target
gene ID Symbol Average site
3656 IRAK2 -2.42 interleukin-1 receptor-associated kinase 2 (-)
3880 KRT19 -2.41 keratin 19 (-)
1978 EIF4EBP1 -2.41 eukaryotic translation initiation factor 4E binding protein 1 (+)
7431 VIM -2.39 vimentin (+)
57568 SIPA1L2 -2.38 signal-induced proliferation-associated 1 like 2 (-)
7913 DEK -2.35 DEK oncogene (+)
123 PLIN2 -2.34 perilipin 2 (-)
4501 MT1X -2.33 metallothionein 1X (-)
654346 LGALS9C -2.31 lectin, galactoside-binding, soluble, 9C (+)
4489 MT1A -2.3 metallothionein 1A (-)
3669 ISG20 -2.28 interferon stimulated exonuclease gene 20 kDa (-)
2920 CXCL2 -2.28 chemokine (C-X-C motif) ligand 2 (-)
2274 FHL2 -2.27 four and a half LIM domains 2 (-)
157506 RDH10 -2.27 retinol dehydrogenase 10 (all-trans) (-)
25937 WWTR1 -2.26 WW domain containing transcription regulator 1 (-)
3690 ITGB3 -2.26 integrin, β 3 (platelet glycoprotein IIIa, antigen CD61) (+)
196513 DCP1B -2.24 DCP1 decapping enzyme homolog B (S. cerevisiae) (-)
9518 GDF15 -2.24 growth differentiation factor 15 (-)
1364 CLDN4 -2.23 claudin 4 (-)
23643 LY96 -2.2 lymphocyte antigen 96 (-)
10561 IFI44 -2.2 interferon-induced protein 44 (-)
84141 FAM176A -2.19 family with sequence similarity 176, member A (-)
6281 S100A10 -2.17 S100 calcium binding protein A10 (-)
7088 TLE1 -2.17 transducin-like enhancer of split 1 (E(sp1) homolog, Drosophila) (-)
81553 FAM49A -2.17 family with sequence similarity 49, member A (-)
4599 MX1 -2.17 myxovirus (inuenza virus) resistance 1,
interferon-inducible protein p78 (mouse) (-)
6850 SYK -2.17 spleen tyrosine kinase (-)
7364 UGT2B7 -2.17 UDP glucuronosyltransferase 2 family, polypeptide B7 (-)
5366 PMAIP1 -2.17 phorbol-12-myristate-13-acetate-induced protein 1 (-)
50515 CHST11 -2.16 carbohydrate (chondroitin 4) sulfotransferase 11 (+)
2982 GUCY1A3 -2.16 guanylate cyclase 1, soluble, α 3 (+)
6273 S100A2 -2.15 S100 calcium binding protein A2 (+)
54478 FAM64A -2.15 family with sequence similarity 64, member A (-)
3428 IFI16 -2.14 interferon, γ-inducible protein 16 (-)
9615 GDA -2.14 guanine deaminase (-)
7849 PAX8 -2.13 paired box 8 (-)
10550 ARL6IP5 -2.11 ADP-ribosylation-like factor 6 interacting protein 5 (+)
23286 WWC1 -2.1 WW and C2 domain containing 1 (+)
9636 ISG15 -2.08 ISG15 ubiquitin-like modier (+)
896 CCND3 -2.07 cyclin D3 (+)
5329 PLAUR -2.07 plasminogen activator, urokinase receptor (-)
4853 NOTCH2 -2.06 Notch homolog 2 (Drosophila) (+)
55652 SLC48A1 -2.05 solute carrier family 48 (heme transporter), member 1 (+)
23476 BRD4 -2.04 bromodomain containing 4 (+)
2012 EMP1 -2.03 epithelial membrane protein 1 (+)
3429 IFI27 -2.02 interferon, α-inducible protein 27 (-)
79710 MORC4 -2.01 MORC family CW-type zinc nger 4 (-)
80820 EEPD1 -2.01 endonuclease/exonuclease/phosphatase family domain containing 1 (+)
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 805-817, 2012
809
dish), 6-well plates for wound healing assays (20x10
4
cells per
well), in 24-well plates for the mRNA extraction and Matrigel
invasion assays (5x10
4
cells per well) and in 96-well plates for
the XTT assays (3,000 cells per well).
Cell morphology. Cells were transfected with miR138 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 modied
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 miR138-transfected A498 cells in comparison
with the miR-negative control transfectant, as previously
described (23). Briey, 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 uoride
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).
Specic 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 Scientic). 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 signicance 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 rstly
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 miR138 functions
Table III. Patient characteristics of immunohistochemistry.
No. of patients (%)
Total number 67
Age (average) 30-80 (54.4)
Gender
Male 45 (67.2)
Female 22 (32.8)
Pathological tumor stage
pT1 15 (22.4)
pT2 28 (41.8)
pT3 22 (32.8)
pT4 2 (3.0)
Grade
G1 52 (77.6)
G2 14 (20.9)
G3 1 (1.5)
Normal tissue 10
YAMASAKI et al: TUMOR SUPPRESSIVE microRNA-138 IN RENAL CELL CARCINOMA
810
Figure 1. Effect of miR138 transfection on RCC cell lines. (A) The change of morphology of miR‑138 transfectants. A498 and 786-O cells were transfected
with miR138 for 72 h and were then examined by an inverted microscope. (B) miR138 expression in A498 and 786-O cell lines and in normal kidney. miR138
expression levels in A498 and 786-O were signicantly lower than those in normal human kidney RNA. RNU6B was used as an internal control. (C-E) Effect of
miR138 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.
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 805-817, 2012
811
Figure 2. miR‑138 regulates molecular targets in RCC cells. (A) Heatmap derived from ve RCC samples. A total of 99 genes were downregulated less than -2.0-fold
in miR138 transfectants. We checked their mRNA expression levels in RCC by using our previous gene expression analysis of ve 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.
YAMASAKI et al: TUMOR SUPPRESSIVE microRNA-138 IN RENAL CELL CARCINOMA
812
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 miR138 were determined by real-time
RT-PCR. The expression levels of miR‑138 were signicantly
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 signi-
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 83.1.1, 100.0±0.4
and 100.3±0.6, respectively, P<0.0001 (Fig. 1C).
The wound healing assay demonstrated that signicant inhi-
bition of cell migration occurred in the miR138 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 signicantly decreased in the miR138-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 83.7.4, 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 miR138 trans-
fectants compared with miRNA-control transfectants in A498
cells. A total of 99 genes were downregulated in miR138 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 miR138 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 (miR138 transfectants).
The two expression signatures in this study (miR138
transfectants and RCC clinical specimens) revealed that VIM
was a promising putative target gene in miR138 in RCC. Thus,
we focused on the VIM gene and investigated the functional
signicance 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
signicantly 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.
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 805-817, 2012
813
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.
YAMASAKI et al: TUMOR SUPPRESSIVE microRNA-138 IN RENAL CELL CARCINOMA
814
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
miR138 (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, 87.1.9 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 signicantly 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 miR138 were signicantly 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
signicantly higher in RCC than adjacent non-cancerous speci-
mens (clinical RCC specimens, 6.017±0.622, adjacent normal
tissues; 1.310.224, P<0.0001) (Fig. 5B).
The mRNA expression of ≥T2 specimens (n=7) was signi-
cantly higher than that of T1 (n=26) (T1, 5.230±0.607; T2,
8.771.349, 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 signicantly 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 signicant
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 signicant.
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 miR138 in RCC clinical samples were signicantly
downregulated compared with adjacent normal kidney. (B) Expression levels of VIM mRNA in RCC clinical samples were signicantly 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 signicantly
higher compared with T1 RCC samples.
INTERNATIONAL JOURNAL OF ONCOLOGY 41: 805-817, 2012
815
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 miR138 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
miR1382, 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.
YAMASAKI et al: TUMOR SUPPRESSIVE microRNA-138 IN RENAL CELL CARCINOMA
816
Importantly, we found signicant morphologic change in
RCC cell lines (A498 and 786-O) by miR138 transfection.
Furthermore, restoration of miR‑138 signicantly 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 miR138 is important for our
understanding of human RCC invasion and metastasis. Based
on this view, we performed molecular target searches for
miR138 in cancer cells by combining two genome-wide gene
expression studies (miR138 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 miR138 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 miR138 signicantly inhibited both mRNA and protein
expression levels of VIM in RCC cells, suggesting VIM was
regulated by tumor suppressive miR138. 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
miR138 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
miR138 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 miR138 expres-
sion in HCC tissues. Our data of miR‑138 transfectants in
RCC cell lines demonstrated that CCND3 is a putative target
of miR138 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 specic 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 signicantly 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 miR138 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 miR138 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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    • "In our present study, we found augmented steroidogenesis as well as up-regulated vimentin in MLTC-1 and Y1 cell lines after low-dose MBP treatment, and verified the role of vimentin in the MBP-induced progesterone secretion, which suggested that the upregulation of vimentin promoted cholesterol transport then resulting in the accretion of steroidogenesis after MBP treatments. miRNAs have been shown to participate in a variety of tumor genesis, and a growing body of evidence suggested that miRNAs play crucial roles in immune evasion, cell cycle regulation and cancer progression (Yamasaki et al., 2012; Yokoi and Nakajima, 2013). miRNA-200c, usually as a tumor suppressor, was studied in various human tumor types (Su et al., 2015). "
    [Show abstract] [Hide abstract] ABSTRACT: The reproductive toxicity of plasticizer di-n-butyl phthalate (DBP) and its active metabolite monobutyl phthalate (MBP) has been demonstrated in rodents. The objective of this study was to explore roles of vimentin and miRNA-200c in steroidogenesis interfered by MBP. Mouse Leydig tumor cells (MLTC-1) and murine adrenocortical tumor cells (Y1) were employed and exposed to various levels of MBP (10(-7), 10(-6), 10(-5) and 10(-4) M). Steroid hormone production was increased significantly when MLTC-1 and Y1 cells were exposed to MBP at 10(-7) M. Additionally, vimentin and steroidogenic acute regulatory protein (StAR) expressions were upregulated at the same dose. It was found that MBP increased the steroidogenesis by facilitating the cholesterol transfer process by vimentin. In contrast, miRNA-200c expression was depressed at doses of MBP (10(-7) M) in both cells. Moreover, vimentin expression and progesterone production were increased in both MLTC-1 and Y1 cells after miRNA-200c expression was artificially inhibited. These results strongly suggested that MBP raised steroid hormone synthesis via upregulated vimentin by miRNA-200c.
    Full-text · Article · Nov 2015
    • "Vimentin is a class-III intermediate filament that is involved in a programmed cell death process [180]; it is controversial whether vimentin is up-regulalted or down-regulated according to previous studies of NPC [133,134,137,138]. One study demonstrated that the expression levels of vimentin were significantly up-regulated in renal cell cancer tissues compared to adjacent non-cancerous tissues, which could enhance migration and invasion activities of cancer cells [181]. These findings suggest that vimentin is a potential therapeutic target in cancer. "
    [Show abstract] [Hide abstract] ABSTRACT: Although radiotherapy is generally effective in the treatment of major nasopharyngeal carcinoma (NPC), this treatment still makes approximately 20% of patients radioresistant. Therefore, the identification of blood or biopsy biomarkers that can predict the treatment response to radioresistance and that can diagnosis early stages of NPC would be highly useful to improve this situation. Proteomics is widely used in NPC for searching biomarkers and comparing differentially expressed proteins. In this review, an overview of proteomics with different samples related to NPC and common proteomics methods was made. In conclusion, identical proteins are sorted as follows: Keratin is ranked the highest followed by such proteins as annexin, heat shock protein, 14-3-3σ, nm-23 protein, cathepsin, heterogeneous nuclear ribonucleoproteins, enolase, triosephosphate isomerase, stathmin, prohibitin, and vimentin. This ranking indicates that these proteins may be NPC-related proteins and have potential value for further studies.
    Full-text · Article · Jul 2015
    • "Vimentin expression is regulated by many factors, such as microRNA-138, which inhibits migration and invasion by directly targeting vimentin in renal cell carcinoma. In addition, the vimentin gene is also highly methylated in CRC tissues [41,42] . Here, we observed that OTUB1 is a new regulator of vimentin in CRC cell lines. "
    [Show abstract] [Hide abstract] ABSTRACT: OTUB1 (OTU deubiquitinase, ubiquitin aldehyde binding 1) is a deubiquitinating enzyme (DUB) that belongs to the OTU (ovarian tumor) superfamily. The aim of this study was to clarify the role of OTUB1 in colorectal cancer (CRC) and to identify the mechanism underlying its function. Two hundred and sixty CRC samples were subjected to association analysis of OTUB1 expression and clinicopathological variables using immunohistochemical (IHC) staining. Overexpression of OTUB1 was achieved in SW480 and DLD-1 cells, and downregulation of OTUB1 was employed in SW620 cells. Then, migration and invasion assays were performed, and markers of the epithelial-mesenchymal transition (EMT) were analyzed. In addition, hepatic metastasis models in mice were used to validate the function of OTUB1 in vivo. OTUB1 was overexpressed in CRC tissues, and the expression level of OTUB1 was associated with metastasis. A high expression level of OTUB1 was also associated with poor survival, and OTUB1 served as an independent prognostic factor in multivariate analysis. OTUB1 also promoted the metastasis of CRC cell lines in vitro and in vivo by regulating EMT. OTUB1 promotes CRC metastasis by facilitating EMT and acts as a potential distant metastasis marker and prognostic factor in CRC. Targeting OTUB1 may be helpful for the treatment of CRC.
    Full-text · Article · Nov 2014
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