Carcinogenesis vol.33 no.3 pp.501–508, 2012
Advance Access publication December 17, 2011
MicroRNA-1826 directly targets beta-catenin (CTNNB1) and MEK1 (MAP2K1) in
VHL-inactivated renal cancer
Hiroshi Hirata, Yuji Hinoda1, Koji Ueno,
Koichi Nakajima2, Nobuhisa Ishii2and Rajvir Dahiya?
Department of Urology, San Francisco Veterans Affairs Medical Center and
University of California at San Francisco, San Francisco, CA 94121, USA,
1Department of Oncology and Laboratory Medicine, Yamaguchi University
GraduateSchool ofMedicine, Yamaguchi 7558505, Japan and2Departmentof
Urology, Toho University Faculty of Medicine, Tokyo 143-8540, Japan
?To whom correspondence should be addressed. Urology Research Center
(112F), Department of Urology, Veterans Affairs Medical Center and
University of California at San Francisco, 4150 Clement Street, San Francisco,
CA 94121, USA.
Tel: þ1 415 750 6964; Fax: þ1 415 750 6639;
The aim of this project is to identify new therapeutic microRNAs
(miRNAs) for von Hippel-Lindau (VHL)-inactivated renal cancer
cells. We initially identified several potential miRNAs targeting
CTNNB1 and MEK1 using several targets scan algorithms. Only
miR-1826 was found to target CTNNB1 and MEK1. Therefore,
we focused onmiRNA-1826 and performed3# untranslatedregion
(UTR) luciferase assay, functional analyses and association study
between miR-1826 expression and renal cancer patient outcomes.
miR-1826 expression was significantly lower in renal cancer tis-
sues compared with non-neoplastic areas and lower expression
was significantly associated with overall shorter survival and ear-
lier recurrence after radical nephrectomy. Following miR-1826
transfection, 3# UTR luciferase activity and protein expression
of beta-catenin and MEK1 were significantly downregulated
in renal cancer cells. Introduction of miR-1826 also inhibited
renal cancer cell proliferation, invasion and migration. Addition-
ally, miR-1826 promoted apoptosis and G1 arrest in VHL-
inactivated renal cancer cells. Knockdowns of CTNNB1 and
effect of miR-1826. Our data suggest that the miR-1826 plays an
important role as a tumor suppressor by downregulating beta-
catenin and MEK1 in VHL-inactivated renal cancers.
Renal cell carcinoma (RCC) is the third leading cause of death among
urological tumors, accounting for 2% of adult malignancies (1).
Although the rate of detection of incidental RCC has increased with
improved diagnostic techniques, metastatic lesions are still found at
diagnosis in ?30% of RCC patients (2). Renal cancer patients with
localized kidney cancer have a 90% 5 years survival rate. However, pa-
survival rate (,30%) (1). Compared with other cancers, there are very
few tumor markers for renal cancer (3). Also, renal cancer patients re-
spond poorly to conventional chemotherapy because RCC is regarded as
a multidrug-resistant cancer (4). Recently, three multikinase inhibitors,
of advanced RCC but are not globally used (5). The most common
histological type of renal cancer is clear cell renal cell carcinoma (cc-
RCC, 70%), and the ‘von Hippel-Lindau’ (VHL) tumor-suppressor
gene is associated with tumorigenesis in cc-RCC (6,7). Approximately
60% of cc-RCC patients have a mutated or inactivated VHL gene. The
main function of VHL protein (p-VHL) is to inhibit beta-catenin
(CTNNB1) and hypoxia inducible factor (HIF)-1 (6–8). Therefore,
mutation of the VHL gene may activate oncogenic pathways, such
as beta-catenin or HIF-1 (6–8). HIF-1 is known to stimulate the Ras–
Raf–MEK–extracellular signal-regulated kinase (ERK) pathway (9).
reported to be important in several cancer treatments (10). miRNAs are
small non-coding RNAs of ?22 nucleotides in length that are capable of
regulating gene expression at both the transcription and translation levels
(11). miRNAs bind to the 3# untranslated region (UTR) of target mes-
senger RNA (mRNA) and repress translation from mRNA to protein or
induce mRNA cleavage and thereby regulate the expression of target
genes (11). Based on several target scan algorithms, we searched for
miRNAs targeting both beta-catenin and HIF-1 and downstream onco-
genes using microRNATarget Prediction and Functional Study Database
(miRDB) (http://mirdb.org/miRDB/) (12) and other target predicting al-
gorithms (microRNA.org and TargetScan). As a result only one, miR-
1826, was found targeting both beta-catenin and MEK1. The role of
MEK1 has been reported in several cancers and MEK inhibitors are also
used for cancer treatment in several cancers (13,14).
Based on these results, we hypothesized that miRNA-1826 may be
hypothesis, we performed 3# UTR luciferase assays to confirm whether
miR-1826 binds to the 3# UTR of these target gene’s mRNAs and af-
fected the function (proliferation, invasion, migration, apoptosis and cell
mRNAs using a small interfering RNA (siRNA) technique to examine
the mechanism of miR-1826 tumor-suppressive function.
Materials and methods
The design and schematic representation of this project
A schematic representation of the role of beta-catenin and MEK1 in VHL-
inactivated renal cancer cells is shown in Figure 1A. The aim of this project is
to identify new therapeutic miRNAs related to VHL-inactivated renal cancer
cells. VHL gene mutation and inactivation results in constitutive HIF activation
and aberrant accumulation of cytoplasmic and nuclear beta-catenin (Figure 1A).
and found several miRNAs targeting beta-catenin and HIF downstream genes
(MEK1/2 and ERK1/2). The miRNAs identified are shown in Figure 1B.
A total of 46 patients (31 male and 15 female) with pathologically confirmed
conventional RCC were enrolled in this study (Toho University Hospital,
Tokyo, Japan). The mean age of the patients was 60.8 years (range 37–77
years). They were classified according to the World Health Organization cri-
teria and staged according to the tumor-node-metastasis (TNM) classification.
NamelyTrefers to the sizeof the renalcancer andwhetheror notithas invaded
nearby tissue, N refers to whether regional lymph nodes are involved and M
whether there is distant metastasis or not. The pathology of all the patients was
cc-RCC. Samples were obtained from the patients after written informed
consent was obtained in Toho University hospital.
Renal cancer cell lines with inactivated VHL [A-498, American Type Culture
Collection (ATCC) number: HTB-44; 786-O, ATCC number: CRL-1932] and
with intact VHL (Caki-1, ATCC number: HTB-46) were purchased from the
ATCC (Manassas, VA). The cell lines were cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum.
Total RNA and protein extraction
RNA (miRNA and total RNA) was extracted from formalin-fixed paraffin-
embedded human renal cancer and matched adjacent non-cancerous normal
kidney tissues using a miRNeasy formalin-fixed paraffin-embedded kit (Qia-
gen) after microdissection. To digest DNA, the Qiagen RNase-Free DNase kit
was used. Total RNA was also extracted from renal cancer cell lines using an
Abbreviations: ATCC, American Type Culture Collection; cc-RCC, clear cell
renal cell carcinoma; ERK, extracellular signal-regulated kinase; HIF, hypoxia
inducible factor; miRDB, microRNA Target Prediction and Functional Study
Database; miRNA, microRNA; mRNA, messenger RNA; PCR, polymerase
chain reaction; RCC, renal cell carcinoma; siRNA, small interfering RNAs;
UTR, untranslated region; VHL, von Hippel-Lindau.
? The Author 2011. Published by Oxford University Press. All rights reserved. For Permissions, please email: firstname.lastname@example.org
miRNeasy mini kit (Qiagen). Cells were lysed with RIPA buffer (Pierce,
Brebieres, France) containing protease inhibitors (Sigma, St Louis, MO).
Protein quantification was done using a BCA protein assay kit (Pierce).
Pre-miRTMmiRNA precursors [negative control (miR-NC) (catalog#AM17110)
or hsa-miR-1826 (miR-1826) (catalog#AM17100; Ambion)] were transiently
transfected into renal cancer cells by Lipofectamine 2000 (Invitrogen) according
to the manufacturer’s instructions.
Cell viability, cell invasion and wound healing assay
Cell viability was measured with CellTiter 96 AQueous One Solution Cell Pro-
liferation Assay (MTS)(Promega) 3 days after transfection. Data are the mean ±
SD of six independent experiments. Cell invasion assay was performed with the
CytoSelect 24-well cell invasion assay kit (Cell BioLab, San Diego, CA) as
described previously (15). Transfected cells (miR-NC or miR-1826 transfec-
tant—48 h)were resuspendedin culture medium and put into the upper chamber
in triplicate. After 48 h incubation at 37?C (5% CO2), cells migrating through
the membrane were stained. The results were expressed as invaded cells quan-
tified at optical density 560 nm. The wound healing process begins with tissue
matrixremodeling,migrationandeventualclosing of thewoundarea.Therefore,
this assay is frequently used for assessment of cancer cell migration. Wound
healing assay was performed with the CytoSelect 24-well wound healing assay
kit as described previously (Cell BioLab)) (15). To generate a wound field,
transfected cells (miR-NC or miR-1826 transfectants—48 h) were cultured until
they formed a monolayer around the insert (24 h). After removing insert, a 0.9
mm open-wound field was generated and cells were allowed to migrate from
either side ofthe gap. Wound closurewas monitored and the percent closurewas
measured at10h using miR-NC andmiR-1826transfectedcells [Percentclosure
rate (%) 5 migrated cell surface area/(total surface area ? 100)].
Apoptosis and cell cycle analyses
washedtwicewith1? phosphate-buffered saline and trypsinized. After inactivat-
buffer (70 ll). Annexin V-fluorescein isothiocyanate solution (10 ll) and
7-aminoactinomycin D viability dye (20 ll) were added to 70 ll of the cell sus-
pensions. After incubation for 15 min in the dark, 400 ll of ice-cold 1? binding
buffer was added. The apoptotic distribution of the cells in each sample was then
determined using a fluorescence-activated cell sorting (Cell Lab QUANTA SC;
Beckman Coulter, Fullerton, CA). The various phases of cells were determined
using a DNA stain (4#,6-diamidino-2-phenylindole). Cell populations (G0/G1, S
and G2/M) were differentiated according to 4#,6-diamidino-2-phenylindole in-
tensity and side scatter-measured cell volume and quantified with Cell Lab
QUANTA software. Data are the mean ± SD of four independent experiments.
3# UTR luciferase assay
To identify potential miRNAs targeting beta-catenin (CTNNB1) and genes
related to the Ras–Raf–MEK–ERK pathway, we used several algorithms
[TargetScan (http://www.targetscan.org/), microRNA.org (http://www.microrna.
org/microrna/home.do/) and miRDB (http://mirdb.org/miRDB/)] (12). Based
on these algorithms, we found several miRNAs targeting CTNNB1 (11 miR-
NAs), MEK1 (23 miRNAs), MEK2 (two miRNAs), ERK1 (three miRNAs) and
ERK2 (26 miRNAs) (Figure 1B). Among these miRNAs, miR-1826 only tar-
gets both CTNNB1 and MEK1 genes. Therefore, we focused on miR-1826 in
this project. In order to perform 3# UTR luciferase assay, PmirGLO Dual-
Luciferase miRNA Target Expression Vector was used (Promega, Madison,
WI). The oligonucleotides sequences (wild-type) used are shown in Supple-
mentary Table S1, available at Carcinogenesis Online. We also constructed
mutated oligonucleotides for each of the wild-type oligonucleotides [mutated-
type, Supplementary Table S2 is available at Carcinogenesis Online]. In a total
volume of 20 ll, 5 ll of 100 lM forward oligonucleotide, 5 ll of 100 lM
reverse oligonucleotide, 2 ll of 10? annealing buffer (100 mM Tris–HCl, pH
7.5, 1 M NaCl and 10 mM ethylenediaminetetraacetic acid) and 8 ll water were
added to a 200 ll polymerase chain reaction (PCR) tube and incubated at 95?C
for 5 min and then placed at room temperature for 1 h. The oligonucleotides
were ligated into the PmeI–XbaI site of pmirGLO Dual-Luciferase miRNA
Fig. 1. Schematic representation of the role of beta-catenin and MEK1 in VHL-inactivated renal cancer cells and potential miRNAs targeting beta-catenin and
MEK1 based on miRDB. (A) When the VHL gene is mutated or inactivated, protein VHL (p-VHL) fails to regulate HIF-1 and beta-catenin, resulting in
constitutive HIF activation and aberrant accumulation of cytoplasmic and nuclear beta-catenin. (B). Based on miRDB, several miRNAs targeting beta-catenin and
HIF downstream genes (MEK1/2 and ERK1/2) were identified.
H.Hirata et al.
Target Expression Vector. Colony direct PCR was performed for insert recogni-
tion using REDTaq (Sigma). The primers used were as follows: forward primer,
5#-cgtgctggaacacggtaaaa-3#; reverse primer, 5#-gcagccaactcagcttcctt-3#. PCR
parameters for cycling were as follows: 94?C for 3 min, 30 cycles of PCR at
94?C for 30 s, 55?C for 30 s, 72?C for 30 s, 72?C for 10 min and 4?C for 10 min.
The PCR product was digested with NotI (TaKaRa/Fisher Scientific, Pittsburgh,
PA). The size of vectors containing inserts were ?200 and 100 bp by electro-
phoresis since the NotI recognition sequence was incorporated into the primers.
Vectors were sequenced directly by an outside vendor (MCLAB, South San
Francisco, CA). For 3# UTR luciferase assay, A-498 and 786-O cells were
cotransfected with miR-NC or miR-1826 and pmirGLO Dual-Luciferase miR-
NATarget Expression Vectors using Lipofectamine 2000 (Invitrogen). Lucifer-
ase assay was performed using the Dual-Luciferase? Reporter Assay System
(Promega) at 48 h after transfection.
Knockdown of CTNNB1 and MEK1 mRNAs in A-498 cells and functional
VHL gene-inactivated A-498 cells were transiently transfected with CTNNB1
and MAP2K1 siRNAs (si-CTNNB1 and si-MAP2K1, catalog#VHS50819,
#VHS40795; Invitrogen, Carlsbad, CA) or negative control siRNA (si-NC;
Invitrogen) according to the manufacturer’s instructions. Briefly, cells were
grown in six-well plates and transfected individually with si-NC (negative
control) or si-CTNNB1 or si-MAP2K1 at a concentration of 200 pmol/well.
Transfection was performed with X-tremeGene siRNA transfection reagent
(Roche Diagnosis, Basel, Switzerland). Then, 48 h after transfection, RNA
and protein were extracted and knockdown of CTNNB1 and MAP2K1 mRNAs
and proteins were confirmed by real-time PCR and western blot analysis. Cell
viability (24, 48 and 72 h after transfection), invasion (48 h after transfection)
and apoptosis analysis (48 h after transfection) were performed using si-NC- or
si-CTNNB1- or si-MAP2K1-transfected A-498 cells.
Quantitative real-time reverse transcription–PCR
Quantitative real-time reverse transcription–PCR was performed in triplicate
with an Applied Biosystems Prism 7500 Fast Sequence Detection System using
TaqMan universal PCR master mix according to the manufacturer’s protocol
(Applied Biosystems, Foster City, CA). The TaqMan probes and primers were
purchased from Applied Biosystems. RNU48 was used as internal control.
Levels of RNA expression were determined using the 7500 Fast System SDS
software version 1.3.1 (Applied Biosystems).
Total cell protein (15 lg) was used for western blotting. Samples were resolved
in 4–20% Precise Protein Gels (Pierce) and transferred to polyvinylidene di-
fluoride membranes (Amersham Biosciences, Fairfield, CT). The membranes
were immersed in 0.3% skim milk in Tris-buffered saline containing 0.1%
Tween 20 for 1 h and probed with primary polyclonal and monoclonal antibody
against beta-catenin (#9562; Cell signaling Technology, Beverly, MA), survivin
(#2808; Cell signaling Technology), VHL (#2738; Cell signaling Technology),
beta-tubulin (#2128; Cell signaling Technology) and MEK1 (#ab47422;
Cambridge, UK) overnight at 4?C. Blots were washed in Tris-buffered saline
containing 0.1% Tween 20 and labeled with horseradish peroxidase-conjugated
secondary anti-mouse or anti-rabbit antibody (Cell signaling Technology).
Proteins were enhanced by chemiluminescence (Amersham ECL plus Western
Blotting detection system; Amersham Biosciences) for visualization. The
protein expression levels were expressed relative to beta-tubulin levels.
Immunohistochemical staining was performed following the manufacturer’s
instructions (UltraVision Detection System; Thermo Fisher Scientific, Fremont,
CA). Namely, formalin-fixed paraffin-embedded tissue sections were incubated
in xylene at room temperature (10 min, three times), rinsed in an ascending
series of ethanol (100, 100, 95 and 70% for 5 min each) and rinsed in ddH2O
for 5 min (high power level) in 1? citrate buffer (pH 6.0) (#21545; Chemicon
endogenous peroxidase, slides were incubated in ‘hydrogen peroxidase block’
(included in kit) for 10 min and then primary antibodies (CTNNB1, MEK1 and
isotype controls) were added and incubated at 4?C overnight. The antibody
dilution was 1:100 based on the manufacturer’s recommendation and we used
the following primary antibodies: beta-catenin (#9562; Cell Signaling Technol-
ogy); MEK1(#ab47422;Abcam,Cambridge,UK).Sectionswere alsoincubated
with an appropriate isotype (IgG) matched rabbit antibody as NC (#3900; Cell
Signaling Technology and #ab27478; Abcam, respectively). After overnight
incubation with primary antibody, slides were washed in Tris-buffered saline
buffer and incubated with secondary biotinylated goat anti-rabbit antibody
(10 min, room temperature) and streptavidin peroxidase was added (10 min,
room temperature). Color was developed with diaminobenzidene (40 ll of
diaminobenzidene Plus Chromogen þ 2ml of diaminobenzidene Plus Substrate)
for 5 min at room temperature. The sections were counterstained with hematox-
ylin (American Master Tech Scientific, Lodi, CA). Immunohistochemical staining
was evaluated by visually assessingstaining intensity (0–2) using a microscope
at ?200. All specimens were scored blindly by two observers. We used the
Human Protein Atlas (http://www.proteinatlas.org/) as a reference for immu-
nohistochemistry assessment. The criteria of intensity are as follows: 0, negative
expression; 1þ, weakly positive expression; 2þ, strongly positive expression.
All statistical analyses were performed using StatView (version 5; SAS
Institute, NC). A P-value of ,0.05 was regarded as statistically significant.
Effect of miR-1826 on VHL proficient renal cancer cell (Caki-1)
We looked at the expression and function of miR-1826-transfected
VHL-proficient renal cancer cells (Caki-1) (Supplementary Figure S1
is available at Carcinogenesis Online). The expression of miR-1826
was significantly higher in VHL-proficient renal cancer cell line
(Caki-1) compared with VHL-inactivated renal cancer cell lines
(A-498 and 786-O) (Supplementary Figure S1A is available at Car-
cinogenesis Online). Next, we performed functional analyses with
miR-1826 overexpressing Caki-1 cells (Supplementary Figure S1B is
Caki-1 cells did not show inhibition of proliferation (Supplementary
Figure S1C is available at Carcinogenesis Online), invasion (Supple-
mentary Figure S1D is available at Carcinogenesis Online) and mi-
gration (Supplementary Figure S1E is available at Carcinogenesis
Online). miR-1826 also did not induce apoptosis in VHL-proficient
renal cancer cells (Caki-1; Supplementary Figure S1F is available at
3# UTR luciferase assay and lower protein expression in miR-1826
CTNNB1 mRNA has one potential complimentary binding site with
miR-1826 within its 3# UTR (mirSVR score: ?1.2735; Figure 2A).
MEK1 mRNA has two potential complimentary binding sites with
miR-1826 within its 3# UTR (mirSVR score: ?0.8212 and ?1.1902,
respectively). Based on these results, we performed 3# UTR luciferase
assays and observed that the relative luciferase activity was signifi-
cantly decreased in miR-1826-transfected VHL-inactivated renal can-
cer cells (A-498 and 786-O) (Figure 2B).
With mutated plasmids, there was no significant difference in lucif-
erase activity between controls and miR-1826 transfectants (Figure2B).
These results suggest that CTNNB1 and MEK1 mRNAs are target
oncogenes of miR-1826. CTNNB1 protein expression was also signif-
icantly decreased in miR-1826-transfected cells (Figure 2C). In order
to verify beta-catenin downregulation, we looked at the expression of
survivin, which are T cell-factor/lymphoid-enhancer-factor downstream
effectors. As expected, the protein (survivin) was significantly down-
regulated in miR-1826-transfected cells (Figure 2C). MEK1 protein
expression was also significantly decreased in miR-1826-transfected
renal cancer cells (Figure 2C).
Inverse relationship between miR-1826 and beta-catenin/MEK1
We investigated the relationship between miR-1826 expression and
beta-catenin/MEK1 protein expression levels in clinical samples. We
observed an inverse correlation between miR-1826 expression and
CTNNB1 or MEK1 protein expression in renal cancer tissues
(Figure 2D). Representative pictures of the stainings for beta-catenin,
MEK1 and isotype control in renal cancer tissues and normal kidney
tissues are shown in Supplementary Figure S2, available at Carcino-
genesis Online. The expression of CTNNB1 and MEK1 was weak in
normal kidney tissues.
Expression of beta-catenin and MEK1 in renal cancer cells
The beta-catenin (CTNNB1) and MAP2K1 protein expression levels
were significantly higher in renal cancer cells with inactivated VHL
miR-1826 and Wnt and HIF-1 signaling pathways in RCC
(A-498 and 786-O) compared with those with intact VHL (Caki-1)
(Supplementary Figure S3A is available at Carcinogenesis Online).
All patient pathology was cc-RCC. MEK1 expression in the renal
cancer tissues was classified into two categories based on immuno-
Higher MEK1 expression was associated with higher pathological
stage (pTNM) and shorter overall survival and recurrence interval after
radical nephrectomy(TableI). Multivariateanalysis for overall survival
also showed higher MEK1 expression in renal cancer tissues that was
significantly associated with shorter overall survival (Table II). Further-
more, Kaplan–Meier plots showed inverse correlation between MEK1
expression and renal cancer patient outcomes (Figure 3).
betweenMEK1 expressionlevel andclinical
Relationship between miR-1826 expression level and clinical
The miR-1826 expression levels were compared based on real-time
PCR results. Namely, the miR-1826 expression level was significantly
higher in normal kidney tissues compared with renal cancer tissues
(n 5 46) (Figure 4A). We also split renal cancer patients into two
groups (L for low and H for high) based on median miR-1826 expres-
sion levels as a cutoff point. We investigated the relationship between
miR-1826 expression level (lower or higher in cancer tissues) and
clinical factors, including grade, pathologic tumor classification
(pT), pathologic lymph node status (pN), pathologic metastasis
status (pM) and outcomes (survival and recurrence). There was no
significant association of grade and pTNM with miR-1826 expression
(data not shown), but we found that patients in the lower miR-1826
category had a significantly poor outcome (Figure 4B).
Effect of miRNA-1826 on cell viability, migration, invasion, cell cycle
and apoptosis in VHL-inactivated renal cancer cells
At 48 h after transfection of miR-NC or miR-1826 into renal cancer
cells (A-498 and 786-O), the increased miR-1826 expression level was
verified by real-time PCR (fold change: 51050 and 2513, respectively;
Supplementary Figure S3B is available at Carcinogenesis Online). We
observed a significant decrease in cell viability (Figure 5A), invasion
(Figure 5B) and migration (Figure 5C) in miRNA-1826-transfected
renal cancer cells compared with miR-NC-transfected cells. We found
a significant difference in the number of apoptotic cells between
miR-1826-transfected cells (A-498 and 786-O) and control cells
(Figure 5D). We also found a significant difference in each of the cell
cycle phases (G0/G1, S and G2/M) between miR-1826 transfectants
and controls. Namely, miR-1826 induced a significant G1arrest in
A-498 renal cancer cells (Supplementary Figure S4 is available at
Effect of CTNNB1 and MEK1 mRNA knockdown on renal cancercells
To look at whether miR-1826 exerts its tumor-suppressive function
through CTNNB1 (beta-catenin) and MEK1 (MAP2K1), we knocked
down CTNNB1 and MEK1 (MAP2K1) mRNAs using an siRNA
technique. The knockdown effect was confirmed by measuring mRNA
and protein expression levels. As shown in Supplementary Figure S5A,
available at Carcinogenesis Online, the relative CTNNB1 and MEK1
Fig.2. BindingofmiR-1826to the3#UTRofCTNNB1andMEK1mRNAs,luciferaseassaysandaninversecorrelationbetweenmiR-1826andCTNNB1/MEK1
protein expression in human renal cancer tissues. (A) CTNNB1 and MEK1 3# UTR position and complementary miR-1826-binding sequence. (B) 3# UTR
luciferase assay (miR-NC and miR-1826), MT stands for mutated plasmid sequence and WT stands for wild-type plasmid sequence. (C) CTNNB1, survivin,
MEK1, beta-tubulin protein expression in miR-NC or miR-1826-transfected VHL-inactivated renal cancer cells (A-498 and 786-O). (D) Inverse correlation
between miR-1826 and CTNNB1/MEK1 protein expression in renal cancer tissues.
H.Hirata et al.
protein expression levels were significantly decreased. Then, we exam-
ined MTS and invasion assays in si-NC, si-CTNNB1 and si-MEK1-
transfected A-498cells.AsshowninSupplementaryFigureS5B and C,
available at Carcinogenesis Online, cell viability (days 3 and 4) and
invasion (48 h) were significantly inhibited inboth si-CTNNB1- and si-
G1 phase arrest was inducted and apoptosis was promoted in si-
CTNNB1- and si-MEK1-transfected A-498 renal cancer cells (Supple-
mentary Figure S5D and E is available at Carcinogenesis Online).
cc-RCC is the most common type in sporadic renal cancer and ?60%
of cc-RCC patients have mutated or inactivated VHL genes. Usually,
pVHL inhibits the HIF-1 and beta-catenin signaling pathway. Thus,
loss of pVHL results in the aberrant accumulation of cytoplasmic and
nuclear beta-catenin and HIF, accelerating tumorigenesis (6,7).
CTNNB1 (beta-catenin) has been known as a key player in the
Wnt/beta-catenin signaling pathway in several cancers including renal
cancer (16). MEK1 (MAP2K1) acts as a mitogen-activated protein
(MAP kinase kinase) and plays an important role as an essential
component of the MAP kinase signal transduction pathway. MEK1
is also involved in several cellular processes, including proliferation
and transcription regulation in several cancers including renal cancer.
In the present study, we found that aberrant MEK1 expression in
human renal cancer tissues was correlated with higher pathological
stage (pTNM) and shorter overall survival and recurrence interval
after radical nephrectomy.Kaplan–Meier plots also showed an inverse
correlation between MEK1 expression and renal cancer patients’ out-
comes. Several MEK1 inhibitors have been identified and have been
evaluated in phases I, II and III clinical trials in several cancers (17).
The effect of MEK1 inhibitors alone or in combination with others,
such as mammalian target of rapamycin inhibitors or epidermal
growth factor receptor inhibitors has been reported in the treatment
of several cancers, including RCC (18,19).However,problems remain
regarding the side effect and efficacy of these treatments. Thus, it is
important to find new and safe options or approaches to achieve renal
cancer remission. Therefore, we focused on miRNAs as a potential
new treatment strategy. We initially searched for tumor-suppressive
miRNAs inhibiting the two major cancer pathways, including beta-
catenin and HIF-1 downstream genes (MEK1, MEK2, ERK1 and
ERK2) with miRDB. This led to the identification of miR-1826 that
only targets beta-catenin and MEK1.
Regarding the relationship between miRNA and CTNNB1, Xia
et al. (20) has previously reported that miR-200a functions as a tumor
suppressor by directly regulating CTNNB1 expression in nasopharyn-
geal carcinoma. Saydam et al. (21) found that miR-200a plays an
important role as a tumor suppressor and directly targets CTNNB1
mRNA. They also showed that miR-200a blocks Wnt/beta-catenin
signaling in meningioma cells (21). However, there have been no
reports related to the role of miRNA and CTNNB1 in renal cancer.
Similarly, miRNAs studies involving MEK1 have found that miR-
34a inhibits cell proliferation by repressing MEK1 during megakar-
yocytic differentiation (22). Also miR-424 regulates cell proliferation
via the silencing of MEK1 and cyclin E1 in senile hemangioma (23).
As far as we know, there have been no reports concerning the relation-
ship of miRNA, MEK1 and renal cancer.
Only one miRNA-1826 was found to target both CTNNB1 and
MEK1. This was also shown by 3# UTR luciferase assays, indicating
that relative luciferase levels were significantly lower in miR-1826-
transfected renal cancer cells compared with miR-NC-transfected
Table I. Relationship between MEK1 (MAP2K1) expression in human
renal cancer tissues and clinico-pathological data
N 5 33 (%)
N 5 13 (%)
Age (mean ± SD) (years)
Male (n 5 31)
Female (n 5 15)
1 (n 5 13)
2 (n 5 28)
3 (n 5 5)
1 (n 5 27)
2 (n 5 10)
1 þ 2 (n 5 37)
3 (n 5 4)
4 (n 5 5)
3 þ 4 (n 5 9)
1 (n 5 27)
2 (n 5 11)
1 þ 2 (n 5 38)
3 (n 5 7)
4 (n 5 1)
3 þ 4 (n 5 8)
(?) (n 5 43)
(þ) (n 5 3)
(?) (n 5 43)
(þ) (n 5 3)
Alive (n 5 38)
Dead (n 5 8)
No (n 5 36)
Yes (n 5 10)
60.1 ± 8.9 62.8 ± 9.80.37
Table II. Association of clinical parameters, MEK1 expression and overall survival
Coef. Standard error Chi-squareP-valueExp. (Coef.) 95% CI
Univariate analysis variables
Grade (3 versus 1 þ 2)
Gender (male versus female)
Age (younger versus older)
pT (pT3 þ pT4 versus pT1 þ pT2)
pN (N1 versus N0)
pM (M1 versus M0)
MEK1 IHC (2 versus 0 þ 1)
Multivariate analysis variables
pT (pT3 þ pT4 versus pT1 þ pT2)
MEK1 IHC (2 versus 0 þ 1)
CI, confidence interval; Coef, coefficient; Exp. (Coef.), e(Coefficient).
miR-1826 and Wnt and HIF-1 signaling pathways in RCC
controls. Though mRNA expression of CTNNB1 and MEK1 was not
changed in miR-1826-transfected cells (data not shown), the protein
expression of CTNNB1 and MEK1 was significantly downregulated
compared with miR-NC-transfected cells. Our results are consistent
with the fact that miRNAs can bind to the 3# UTR of target mRNA
and repress translation from mRNA to protein (11). Thus, our results
suggest that CTNNB1 and MEK1 are direct targets of miR-1826. We
also performed an immunohistochemical study of CTNNB1 and
MEK1 to examine the relationship between miR-1826 and CTNNB1
or MEK1 expression levels in human renal cancer tissues. We found
an inverse correlation between miR1826 and CTNNB1 or MEK1
During renal cancer progression, beta-catenin (CTNNB1) is translo-
cated into the nucleus, binds to T cell-factor/lymphoid-enhancer-factor
ber of the T cell-factor/lymphoid-enhancer-factor downstream effectors,
to assess whether miR-1826 inhibits downstream Wnt/beta-catenin sig-
naling by beta-catenin downregulation. We found that expression of
survivin was also downregulated by miR-1826 in transfected cells.
Fig. 4. miR-1826 expression level in normal kidney and matched renal cancer tissues and the relationship between miR-1826 expression and renal cancer patient
outcomes. (A) miR-1826 expression level was significantly lower in renal cancer tissues compared with matched normal kidney tissues (n 5 46; cc-RCC). (B-1)
Inverse correlation between miR-1826 expression and overall survival. (B-2) Inverse correlation between miR-1826 and recurrence-free survival after radical
Fig. 3. Inverse correlation between MEK1 expression and RCC patient outcomes. (A) Inverse correlation between MEK1 protein expression and overall survival
after radical nephrectomy. (B). Inverse correlation between MEK1 protein expression and recurrence-free survival after radical nephrectomy.
H.Hirata et al.
other laboratories have reported that the expression of survivin is asso-
ciated with renal cancer aggressiveness and an important prognostic
marker for renal cancer (26,27). Although other factors, such as insu-
lin-like growth factor 1, interferon and nuclear factor-kappaB also reg-
important inhibitor of survivin in cc-RCC.
As a next step, we found that the expression of miR-1826 was
significantly downregulated in renal cancer tissues compared with
matched normal kidney tissues (n 5 46). These results are consistent
with those of miR-1826 expression in normal kidney and renal cancer
cell lines, suggesting that miR-1826 may have tumor-suppressive func-
tions in renal cancer. We did find a significantly shorter overall survival
and earlier recurrence after radical nephrectomy in patients with ‘lower
expression of miR-1826’. This result suggests that low miR-1826 ex-
pression in renal cancer tissues may contribute to poor patient progno-
sis. However, our sample number is relatively small, a larger study will
be needed to look at the correlation between miR-1826 expression and
clinical parameters. We also performed several functional assays using
miR-1826 or miR-NC-transfected VHL-inactivated renal cancer cells
(A-498 and 786-O). As expected, we found that overexpression of
miR-1826 significantly inhibited renal cancer cell proliferation and
also significantly inhibited renal cancer cell migration and invasion
abilities. We also found that miR-1826 induced significant G1cell
cycle arrest and apoptosis in VHL-inactivated renal cancer cells. To
CTNNB1 and MEK1 expression, we performed functional analysesof
CTNNB1 and MEK1 in A-498 cells using a siRNA technique. The
knockdown of CTNNB1 and MEK1 was confirmed at the mRNA
(data not shown) and protein levels. We, then performed functional
analyses (MTS and invasion analyses) and found an effect similar
to that of miR-1826 overexpression. Namely, CTNNB1 or MEK1
knockdown resulted in inhibition of renal cancer cell proliferation
and invasion ability. Taken together, this evidence suggests that
miR-1826 exerts its tumor-suppressive effects through beta-catenin
and MEK1 downregulation in renal cancer cells.
In conclusion, this is the first report documenting that miR-1826
expression is significantly decreased in human renal cancer tissues
where it functions as a tumor suppressor by inhibiting CTNNB1 and
MEK1 expression. Our results suggest that miR-1826 may play a ther-
apeutically important role in renal cancer patients, especially in those
with VHL-inactivated renal cancer.
Supplementary Tables S1 and S2 and Figures S1–S5 can be found at
This study was supported by grants (RO1CA130860, RO1CA160079,
RO1CA138642, T32-DK07790, 1I01BX001123) from the National
Fig. 5. Effect of microR-1826 overexpression on VHL-inactivated renal cancer cells (A-498 and 786-O). (A) Cell viability assay (miR-NC or miR-1826-
transfected A-498 cells and 786-O cells), (B) Invasion assay, (C) Wound healing assay, (D) Flow cytometry analysis of apoptosis in miR-NC or miR-1826-
transfected A-498 cells. Data are the mean ± SD of four independent experiments.
miR-1826 and Wnt and HIF-1 signaling pathways in RCC
Institutes of Health, VA Research Enhancement Award Program
(REAP), Merit Review grants and Yamada Science Foundation.
We thankDr Roger Ericksonforhissupportandassistancewiththe preparation
of the manuscript.
Conflict of Interest Statement: None declared.
1.Jemal,A. et al. (2008) Cancer statistics, 2008. CA Cancer J. Clin., 58,
2.Bukowski,R.M. (1997) Natural history and therapy of metastatic renal cell
carcinoma: the role of interleukin-2. Cancer, 80, 1198–1220.
3.Linehan,W.M. et al. (2010) Molecular diagnosis and therapy of kidney
cancer. Annu. Rev. Med., 61, 329–343.
4.Walsh,N. et al. (2009) Expression of multidrug resistance markers ABCB1
(MDR-1/P-gp) and ABCC1 (MRP-1) in renal cell carcinoma. BMC Urol.,
5.Pirrotta,M.T. et al. (2011) Targeted-therapy in advanced renal cell carci-
noma. Curr. Med. Chem., 18, 1651–1657.
6.Linehan,W.M. et al. (2009) VHL loss of function and its impact on onco-
genic signaling networks in clear cell renal cell carcinoma. Int. J. Biochem.
Cell Biol., 41, 753–756.
7.Linehan,W.M. et al. (2007) Identification of the genes for kidney cancer:
opportunity for disease-specific targeted therapeutics. Clin. Cancer Res.,
8.Peruzzi,B. et al. (2006) The von Hippel-Lindau tumor suppressor gene
product represses oncogenic beta-catenin signaling in renal carcinoma
cells. Proc. Natl Acad. Sci. USA, 103, 14531–14536.
9.Conrad,P.W. et al. (1999) EPAS1 trans-activation during hypoxia requires
p42/p44 MAPK. J. Biol. Chem., 274, 33709–33713.
10.Trang,P. et al. (2008) MicroRNAs as potential cancer therapeutics. Onco-
gene, 27, S52–S57.
11.Inui,M. et al. (2010) MicroRNA control of signal transduction. Nat. Rev.
Mol. Cell Biol., 11, 252–263.
12.Wang,X. (2008) miRDB: a microRNA target prediction and functional
annotation database with a wiki interface. RNA, 14, 1012–1017.
13.Kohno,M. et al. (2006) Targeting the ERK signaling pathway in cancer
therapy. Ann. Med., 38, 200–211.
14.Yacoub,A. et al. (2006) MEK1/2 inhibition promotes Taxotere lethality in
mammary tumors in vivo. Cancer Biol. Ther., 5, 1332–1339.
15.Hirata,H. et al. (2011) DICKKOPF-4 activates the noncanonical c-Jun-NH2
kinase signaling pathway while inhibiting the Wnt-canonical pathway in
human renal cell carcinoma. Cancer, 117, 1649–1660.
16.Barker,N. et al. (2006) Mining the Wnt pathway for cancer therapeutics.
Nat. Rev. Drug Discov., 5, 997–1014.
17.Yamaguchi,T. et al. (2011) Antitumor activities of JTP-74057 (GSK1120212),
a novel MEK1/2 inhibitor, on colorectal cancer cell lines in vitro and in vivo.
Int. J. Oncol., 39, 23–31.
18.Costa,L.J. et al. (2007) Upstream signaling inhibition enhances rapamycin
effect on growth of kidney cancer cells. Urology, 69, 596–602.
19.Eulitt,P.J. et al. (2011) Enhancing mda-7/IL-24 therapy in renal carcinoma
cells by inhibiting multiple protective signaling pathways using sorafenib
and by Ad.5/3 gene delivery. Cancer Biol. Ther., 10, 1290–1305.
20.Xia,H. et al. (2010) miR-200a-mediated downregulation of ZEB2 and
CTNNB1 differentially inhibits nasopharyngeal carcinoma cell growth,
migration and invasion. Biochem. Biophys. Res. Commun., 391, 535–541.
21.Saydam,O. et al. (2009) Downregulated microRNA-200a in meningiomas
promotes tumor growth by reducing E-cadherin and activating the Wnt/
beta-catenin signaling pathway. Mol. Cell. Biol., 29, 5923–5940.
22.Ichimura,A. et al. (2010) MicroRNA-34a inhibits cell proliferation by re-
pressing mitogen-activated protein kinase kinase 1 during megakaryocytic
differentiation of K562 cells. Mol. Pharmacol., 77, 1016–1024.
23.Nakashima,T. et al. (2010) Down-regulation of mir-424 contributes to the
abnormal angiogenesis via MEK1 and cyclin E1 in senile hemangioma: its
implications to therapy. PLoS One, 5, e14334
24Bilim,V. et al. (2000) Altered expression of beta-catenin in renal cell cancer
and transitional cell cancer with the absence of beta-catenin gene mutations.
Clin. Cancer Res., 6, 460–466.
25Parker,A.S. et al. (2006) High expression levels of survivin protein inde-
pendently predict a poor outcome for patients who undergo surgery for clear
cell renal cell carcinoma. Cancer, 107, 37–45.
26Crispen,P.L. et al. (2008) Predicting disease progression after nephrectomy
for localized renal cell carcinoma: the utility of prognostic models and
molecular biomarkers. Cancer, 113, 450–460.
27Wang,G.C. et al. (2009) Expression of cortactin and survivin in renal cell
28Sato,A. et al. (2006) Survivin associates with cell proliferation in renal
cancer cells: regulation of survivin expression by insulin-like growth fac-
tor-1, interferon-gamma and a novel NF-kappaB inhibitor. Int. J. Oncol., 28,
29Griffith,T.S. et al. (2002) Induction and regulation of tumor necrosis factor-
related apoptosis-inducing ligand/Apo-2 ligand-mediated apoptosis in renal
cell carcinoma. Cancer Res., 62, 3093–3099.
Received July 15, 2011; revised December 6, 2011;
accepted December 11, 2011
H.Hirata et al.