MicroRNA-23b Functions as a Tumor Suppressor by
Regulating Zeb1 in Bladder Cancer
Shahana Majid1*, Altaf A. Dar2, Sharanjot Saini1, Guoren Deng1, Inik Chang1, Kirsten Greene1,
Yuichiro Tanaka1, Rajvir Dahiya1, Soichiro Yamamura1
1Department of Urology, VA Medical Center and UCSF, San Francisco, California, United States of America, 2California Pacific Medical Center Research Institute, San
Francisco, California, United States of America
MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by targeted repression of transcription and
translation. In this study we show that miRNA-23b (miR-23b) acts as a tumor suppressor in bladder cancer. Quantitative real-
time PCR analysis showed that miR-23b is significantly down-regulated in bladder cancer cell lines and tumor tissues
compared to non-malignant cells and normal tissue samples. We also demonstrate that miR-23b expression has a potential
to be diagnostic and prognostic biomarker in bladder cancer. High miR-23b expression is positively correlated with higher
overall survival of bladder cancer patients as revealed by Kaplan-Meier analysis. ROC analysis showed that miR-23b
expression can distinguish between normal and bladder cancer tissues. Further we elucidated the biological significance of
miR-23b in bladder cancer. Over-expression of miR-23b in bladder cancer cells inhibited cell proliferation and impaired
colony formation. Fluorescence activated cell sorting (FACS) analysis revealed that re-expression of miR-23b in bladder
cancer cells induced G0/G1 cell cycle arrest and apoptosis while inhibiting cell migration and invasion. Luciferase reporter
assays demonstrated that Zeb1, a crucial regulator of epithelial-to-mesenchymal transition (EMT), is a direct target of miR-
23b in bladder cancer. These results show that loss of miR-23b confers a proliferative advantage and promotes bladder
cancer cell migration and invasion. Furthermore, re-expression of miR-23b may be a beneficial therapeutic strategy for the
treatment of human bladder cancer.
Citation: Majid S, Dar AA, Saini S, Deng G, Chang I, et al. (2013) MicroRNA-23b Functions as a Tumor Suppressor by Regulating Zeb1 in Bladder Cancer. PLoS
ONE 8(7): e67686. doi:10.1371/journal.pone.0067686
Editor: Karl X. Chai, University of Central Florida, United States of America
Received March 17, 2013; Accepted May 20, 2013; Published July 2, 2013
Copyright: ? 2013 Majid et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by National Center for Research Resources of the National Institutes of Health through grant numbers RO1CA138642,
RO1CA130860, RO1CA160079, I01BX001123, VA Merit Review and VA Program Project. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: SMajid@urology.ucsf.edu
Bladder cancer is the fourth most common malignancy in the
United States and one of the costliest to clinically manage .
More than 90% of urinary bladder tumors are comprised of
transitional cell carcinoma (TCC) that arises from transitional
epithelium . Urinary bladder tumors are classified into two
distinct categories: non-muscle and muscle invasive bladder cancer
[3,4]. Most tumors (75–80%) present as low-grade papillary non-
invasive tumors that rarely progress to become lethal but almost
always recur. This type of cancer is called ‘‘superficial’’ bladder
cancer and requires expensive long-term management. The rest
are high-grade muscle invasive tumors (,15%) that can rapidly
progress to become metastatic and lead to death . Etiological
factors involved in bladder carcinogenesis remain unidentified,
and effective molecular markers for the disease are limited.
MicroRNAs (miRNAs) are small, non-coding RNAs that
regulate gene expression by targeted repression of transcription
and translation. Several studies have done a global analysis of
miRNA expression in human cell lines and found tissue and
disease-specific expression patterns [5,6]. There is also increasing
evidence that miRNA expression profiles may be indicative of
disease risk and burden. Thus, miRNAs are being assessed as
potential biomarkers to aid in the diagnosis and prognosis of
different types of cancers [7,8]. Several human miRNAs have been
shown to be dysregulated in bladder cancer, including miR-1280,
miR-203, miR-125b and miR-133a [9,10,11,12] and contribute to
the development and progression of the disease. Here we report
that miR-23b is significantly down-regulated in bladder cancer
tissues and cell lines and that high expression level of miR-23b
positively correlate with higher overall survival of patients after
surgery. In addition, we examined the functional significance of
miR-23b and identified Zeb1 as a direct target of miR-23b in
bladder cancer. For the first time this study shows that miR-23b is
a potential biomarker and tumor suppressor in bladder cancer
directly targeting oncogene Zeb1.
Materials and Methods
Cell Lines and Cell Culture
SV-HUC-1, T24 and J82 cells were purchased from the
American Type Culture Collection (ATCC) and grown according
to ATCC protocols. These human-derived cell lines were
authenticated by DNA short-tandem repeat analysis by ATCC.
The experiments with cell lines were performed within 6 months
of their procurement/resuscitation. SV-HUC-1 cells were cultured
in F-12K Medium (ATCC) with 10% FBS. T24 cells were
cultured in McCoy’s 5A medium supplemented with 10% FBS
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and J82 cells were cultured in Minimum Essential Media (MEM)
supplemented with 10% FBS. Cells were maintained in an
incubator with a humidified atmosphere of 95% air and 5% CO2
Plasmids, Precursors and Transfection
TaqMan probes and precursors for hsa-miR-23b and negative
control pre-miR were purchased from Applied Biosystems (Foster
City, CA). pmir-GLO Dual-Luciferase miRNA Target Expression
Vector was purchased from Promega. microRNA-23b, control-
microRNA and siRNAs were used at 50 nM concentration and
Lipofectamine 2000 (Invitrogen) was used for all transfections.
miRNA and total RNA were extracted from cell lines using a
miRNeasy Mini Kit and an RNeasy Mini Kit (Qiagen). miRNAs
from clinical samples were extracted using laser capture micro-
dissection techniques with a miRNeasy FFPE kit (Qiagen).
Human Clinical Samples
Clinical samples were obtained from the San Francisco
Veterans Affairs (VA) Medical Center. Written informed consent
was obtained from all patients and the study was approved by the
UCSF Committee on Human Research (Approval number:
Quantitative Real-time PCR
Mature miRNAs were assayed using TaqMan MicroRNA
Assays in accordance with the manufacturer’s instructions
(Applied Biosystems). All RT reactions, including no-template
controls and RT minus controls, were run in a 7500 Fast Real
Time PCR System (Applied Biosystems). RNA concentrations
Figure 1. miR-23b expression profile and antiproliferative effect in bladder cancer. A) Quantitative RT-PCR analysis of miR-23b in cell lines
and in matched laser-captured microdissected tissue samples. B) Proliferation of J82 and T24 cells after miR-23b transfection was significantly
reduced compared to cont-miR. C) miR-23b over-expression significantly inhibits colony forming ability of bladder cancer cells.
miR-23b in Bladder Cancer
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were determined with a NanoDrop (Thermo Scientific, Rockford,
IL). Samples were normalized to RNU48 (Applied Biosystems).
Gene expression levels were quantified using the 7500 Fast Real
Time Sequence detection system Software (Applied Biosystems).
Comparative real-time PCR was performed in triplicate, including
no-template controls. Relative expression was calculated using the
Cell Viability and Clonability Assays
Cell viability was determined at 24, 48 and 72 h by using the
CellTiter 96 AQueous One Solution Cell Proliferation Assay kit
(Promega, Madison, WI) according to the manufacturer’s proto-
col. Absorbance was measured at 490 nm using SpectraMAX 190
(Molecular Devices). Data are presented as the mean value for
triplicate experiments compared to the negative control. For
colony formation assay, cells were seeded at low density (1000
cells/plate) and allowed to grow until visible colonies appeared.
Then, cells were stained with Giemsa and colonies were counted.
Migration and Invasion Assays
Cytoselect 24-well cell migration and invasion assay kits (Cell
Biolabs, Inc) were used for migration and invasion assays
according to the manufacturer’s protocol. Briefly, T24 and J82
cells transfected with Pre-miR miRNA precursor or negative
control were harvested 72 hours after transfection and re-
suspended in serum-free Opti-MEM. Cells (106104per 300 ml
media without serum) were added to the upper chamber, and the
lower chamber was filled with 500 ml of media containing 10%
FBS. Cells were incubated for 16 hours at 37uC in a 5% CO2
incubator. After 16 hours, non-migrated/non-invading cells were
removed from upper side of trans-well membrane filter inserts
using a cotton-tipped swab. Migrated/invaded cells on the lower
side were stained and the absorbance was read at 560 nm
according to the manufacturer’s protocol.
Protein was isolated from confluent (70–80%) plates of cultured
cells using the M-PER Mammalian Protein Extraction Reagent
(Pierce Biotechnology, Rockfield, IL) following the manufacturer’s
directions. Protein concentrations were determined by the
Bradford method. Equal amounts of protein were resolved on
4–20% sodium dodecyl sulfate (SDS) polyacrylamide gels and
transferred to a nitrocellulose membrane by voltage gradient
transfer. The resulting blots were blocked with 5% non-fat dry
milk and probed with specific antibodies. Blots were then
incubated with appropriate peroxidase-conjugated secondary
antibodies and visualized using enhanced chemiluminescence
(Pierce Biotechnology, Rockford, IL).
Depletion of Zeb1 Using Small Interfering RNA (siRNA)
T24 bladder cancer cells were plated 24 hours before
transfection. At 40 to 50% confluence, cells were transfected
using lipofectamine-2000 (Invitrogen, Carlsbad, CA) with siRNA
duplexes specific for human Zeb1 (SR304746; Origene Technol-
ogy, Rockville, MD) or control non-silencing (NS) siRNA for 72
hours. Initially, two different sets of siRNA duplexes were tested to
evaluate the target specificity and knockdown efficiency. One
Figure 2. miR-23b induces cell cycle arrest and apoptosis in bladder cancer cells. A–B) Representative pictures of FACS analysis showing
miR-23b over-expression induces G0/G1 cell cycle arrest in J82 and T24 cells with a corresponding decrease in S-phase cells. C–D) miR-23b over-
expression induces apoptosis in J82 and T24 cells with a concomitant decrease in the viable number of cells. Data is shown from triplicate
miR-23b in Bladder Cancer
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siRNA duplex was used for further experiments at 50 nM
Luciferase Reporter Assay
A pmirGLO Dual-Luciferase miRNA target expression vector
was used for 39-UTR luciferase assays (Promega, Madison, WI).
The target oncogene of miRNA-23b was selected on the basis of
online microRNA target database http://www.microrna.org/
microrna/home.do. The primer sequences for the wild type
39UTR were: Forward59
TATGTTTTTTAAAATGTGAGT 39 and Reverse 59 CTA-
GAGCT 39. For the mutant 39UTR, the primer sequences were:
TAAGGCTCACTT 39 and reverse 59 CTAGAAGTGAGCCT-
TAGTGCGCACGATACTAGCGGCCGCGAGCT 39. For lu-
cifease assay, T24 and J82 cells were cotransfected with hsa-miR-
23b and pmirGLO Dual-Luciferase miRNA target expression
vectors with wild-type or mutant target sequence using Lipofecta-
mine 2000. Firefly luciferase activities were measured using the
Dual Luciferase Assay (Promega, Madison, WI) 18 hr after
transfection and the results were normalized with Renilla
luciferase. Each reporter plasmid was transfected at least three
times (on different days) and each sample was assayed in triplicate.
Statistical analyses were performed with GraphPad Prism 5 and
MedCalc version 10.3.2. All quantified data represents an average
of at least triplicate samples or as indicated. Error bars represent
standard deviation of the mean. All tests were performed two
tailed and p-values ,0.05 were considered statistically significant.
Receiver operating curves (ROC) were calculated to determine the
Figure 3. Ectopic expression of miR-23b inhibits bladder cancer cell migration and invasion. A) Migration assays of J82 and T24 cells
transfected with miR-23b. B) Representative pictures of migration assay. C) Invasion assays show a significant decrease in the number of invading J82
and T24 cells transfected with miR-23b. D) Representative pictures of invasion assay.
miR-23b in Bladder Cancer
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Figure 4. miR-23b directly targets EMT regulator Zeb1. A) Complimentary miR-23b binding sequences in the Zeb1 39UTR. B) Western blot
analysis shows that miR-23b represses translation of Zeb1 protein in J82 and T24 bladder cancer cells. C) Luciferase assays showing decreased
reporter activity after co-transfection of either the wild type or mutant Zeb1-39UTR with miR-23b in J82 and T24 cells. Mut- Mutated Zeb1 39UTR
miR-23b in Bladder Cancer
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potential of miR-23b to discriminate between malignant and non-
malignant samples. For survival analysis, Kaplan-Meier (log-rank
test) analysis was performed.
Figure 5. Depletion of Zeb1 by siRNA mimics miR-23b overexpression. A) Zeb1 protein levels were significantly attenuated with 50 nM
siRNA duplexes (Si) compared to a non-silencing siRNA duplex (Con) in T24 cells. B) Effect on cell proliferation. C–D) Effect on migration and invasion
of T24 bladder cancer cells. E) Apoptosis assay showing induction of apoptosis after Zeb1 knockdown by siRNA in T24 cells. *p,0.05.
miR-23b in Bladder Cancer
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miR-23b Expression is Depleted in Bladder Tumors and
Cancer Cell Lines
Preliminary microRNA microarray data revealed that miR-23b
was highly downregulated in bladder cancer cell lines compared to
the non-malignant SV-HUC1 cell line. We validated the
microarray data by miRNA-quantitative RT-PCR (miR qRT-
PCR) analysis and results confirmed that miR-23b was downreg-
ulated in bladder cancer cell lines J82, T24 compared to non-
malignant cell line SV-HUC1 (Figure 1A). To examine the
biological relevance of miR-23b, its expression was analyzed in
laser captured microdissected (LCM) human bladder tumor tissues
and compared to normal matched control tissues. The expression
Figure 6. Diagnostic and prognostic significance of miR-23b in bladder cancer. A) Clinicopathological characteristics of patient cohort. B)
ROC curve analysis showing ability of miR-23b expression to discriminate between malignant and non-malignant tissue samples. C) Kaplan-Meier
analysis for overall survival based on miR-23b expression.
miR-23b in Bladder Cancer
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of miR-23b was found to be significantly down-regulated in all the
tumor samples compared to their matched normal samples
(Figure 1A). Further the expression of miR-23b in normal tissues
correlated with that of the non-malignant cell line and that of
tumor correlated with the cancer cell lines (Figure 1A) indicating
that these cancer cell lines represent a model system to analyze
miR-23b function in bladder cancer. These results also suggest a
putative tumor suppressor role for miR-23b in bladder cancer.
MicroRNA-23b Regulates Bladder Cancer Cell
Proliferation and Colony Formation
To determine the functional significance of miR-23b over
expression in bladder cancer, we transfected bladder cancer cell
lines J82 and T24 with miR-23b precursors. Ectopic expression of
miR-23b significantly decreased cell proliferation as compared to
cells expressing cont-miR (Figure 1B). miR-23b transfected cells
had low colony formation ability as the number of foci in miR-23b
expressing cells was decreased when compared with cont-miR
transfected cells (Figure 1C). These results indicate anti-prolifer-
ative effect of miR-23b in bladder cancer.
miR-23b Triggers Cell Cycle Arrest and Induces Apoptosis
in Bladder Cancer Cells
FACS (fluorescence activated cell sorting) analysis revealed that
re-expression of miR-23b led to a significant increase in the
number of cells in the G0/G1 phase of the cell cycle (59% to 67%)
while the S-phase population decreased from 18% to 9% in J82
cells (Figure 2A). Similar results were observed in T24 cells with an
increase in the G0/G1 cell population (70% to 84%) and a
decrease in S-phase population (15% to 5%) (Figure 2B). Thus
suggesting that miR-23b triggers G0/G1 arrest in miR-23b
transfected cells compared to cont-miR. FACS analysis for
apoptosis was performed using Annexin-V-FITC-7-AAD dye.
The percentage of total apoptotic cells (early apoptotic+apoptotic)
was significantly increased (4% to 19%) in response to miR-23b
over-expression compared to cont-miR with a corresponding 14%
decrease in the viable cell population in J82 cells (Figure 2C). In
T24 cells, an increase (4% to 9%) in apoptotic cells was observed
with miR-23b over-expression compared to cont-miR (Figure 2D).
These results indicate a tumor suppressor role for miR-23b in
miR-23b Suppresses Bladder Cancer Cell Migration and
Over-expression of miR-23b had anti-migratory and anti-
invasive effects on bladder cancer cell lines. Less absorbance was
observed at 560 nm with miR-23b transfected bladder cancer cells
compared to cont-miR in the migration assay (Figure 3A) and
miR-23b over-expression also significantly reduced the invasive-
ness of bladder cancer cells (Figure 3B).
Oncogene Zeb1 is a Direct Target of miR-23b
Zeb1 has been reported to be an important molecule that drives
bladder cancer cell motility. Using an online microRNA target
database we found oncogene Zeb1 to be a potential target of miR-
23b with a complementary 39UTR binding site for the seed
sequence of miR-23b (Figure 4A). We performed Western analysis
with miR-23b transfected cells and found that miR-23b attenuated
expression of Zeb1 protein compared to cont-miR in both J82 and
T24 bladder cancer cells (Figure 4B). To check whether a direct
interaction is involved between miR-23b and its target oncogene
Zeb1, we performed luciferase reporter assays. We found that co-
transfection of miR-23b along with the wild type 39UTR of Zeb1
caused a significant decrease in luciferase activity compared to
controls (Figure 4C). These results suggest that miR-23b directly
targets oncogene Zeb1.
Depletion of Zeb1 by RNA Interference Mimics miR-23b
Reconstitution in Bladder Cancer
Phenocopy experiments were also performed by siRNA
inhibition of Zeb1 (Figure 5). We validated two sets of siRNA
(Si-1 and Si-2) that resulted in significant knockdown of Zeb1 at
protein level (Figure 5A) in T24 bladder cancer cells and used one
siRNA duplex for further experiments at 50 nM concentration.
Our results showed that siRNA inhibition of Zeb1 caused
decreased cell viability (Figure 5B), migratory and invasive
capability (Figure 5C–D) of T24 cancer cells. We also observed
that siRNA inhibition of Zeb1 increased approximately 7% of the
apoptotic fraction of cells in Zeb1 siRNA transfected cells
compared to ,1% in non-specific control (Figure 5E). These
results suggest that siRNA depletion of Zeb1 mimics the effect of
miR-23b over-expression in bladder cancer.
Diagnostic and Prognostic Significance of miR-23b in
To determine whether miR-23b expression can discriminate
between bladder tumors and normal tissues, and predict patient
survival, we performed ROC analysis and Kaplan-Meier analysis.
The clinical demographics of the patient cohort are summarized in
Figure 6A. The area under the ROC curve (AUC) of 0.885
(P,0.0001; 95% CI=0.75 to 0.97) (Figure 6B) suggested that
miR-23b expression can discriminate between malignant and non-
malignant tissues and potentially be used as a diagnostic marker
for bladder cancer. To determine whether miR-23b has any
prognostic significance, we divided patient tissues into low
(expression T/N,1.2 fold) and high (expression T/N.1.2 fold)
miR-23b groups and performed Kaplan-Meier survival analysis.
Kaplan-Meier analysis showed that the high miR-23b group had
significantly higher overall survival probability compared to the
low miR-23b group (Logrank Test p,0.03, Hazard Ratio
(HR)=4.3, 95%CI=2–14) (Figure 6C). These findings suggest
that miR-23b is potentially a diagnostic and prognostic marker for
bladder cancer though studies on additional samples are needed to
strengthen these results.
MicroRNAs can have large-scale effects by regulating expres-
sion of a variety of genes during mammalian development and
carcinogenesis. As a result, understanding the mechanisms and
function of individual miRNAs has generated great interest.
Despite the accumulating evidence regarding the role of various
miRNAs in cancer, very limited information is available about the
function of miRNAs in bladder cancer, and only few miRNA
targets have been identified.
Here we report that miR-23b to be down-regulated in bladder
cancer tissues compared to normal adjacent tissues and this was
also observed in bladder cancer and non-malignant cell lines. Our
data suggests a potential diagnostic/prognostic role for miR-23b in
predicting overall survival and discriminating malignant from
normal tissues and indicates that miR-23b is a tumor suppressor in
To determine the biological relevance of miR-23b in bladder
cancer, we performed functional assays. Ectopic expression of
miR-23b resulted in significant inhibition of cell proliferation,
colony formation, migration/invasion and induction of cell cycle
arrest and apoptosis in bladder cancer cells. Expression of miR-
miR-23b in Bladder Cancer
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23b in cancer is somewhat controversial because it has been found
to be either up-regulated and oncogenic in kidney cancer where it
caused translational repression of tumor suppressor PTEN gene
 or down-regulated and a tumor suppressor in prostate cancer
where it directly targets Src kinase and Akt oncogenes , while
our study indicates it is a tumor suppressor in bladder cancer.
Previous studies have shown that microRNAs are highly tissue
specific and they can act as tumor suppressor or oncogenes
[15,16]. MicroRNAs possess several features that make them
attractive candidates as new prognostic biomarkers and powerful
tools for the early diagnosis of cancer . In this study, we found
that miR-23b was predictive of overall survival such that patients
with higher miR-23b expression had longer overall survival
compared to patients with low miR-23b expression. MicroRNA-
23b expression was also able to distinguish malignant from normal
tissues indicating the diagnostic significance of miR-23b in bladder
cancer although additional studies with a larger cohort of tissue
samples are required.
A significant obstacle to understanding miRNA function has
been the relative paucity of experimentally validated targets. To
determine the effectors of miR-23b, in-silico algorithms and
functional analyses identified Zeb1 as its target. We demonstrated
that miR-23b directly targets the 39UTR of Zeb1, as its over-
expression was associated with suppression of luciferase activity. In
addition, a significant down-regulation in the level of Zeb1 protein
was observed after miR-23b over-expression, indicating post-
transcriptional regulation of Zeb1 via targeting its 39UTR.
Functional assays performed after Zeb1 depletion by siRNA
transfection mimicked the results obtained with miR-23b overex-
pression. These results indicate that effects of miR-23b in bladder
cancer are partly by directly targeting Zeb1, though other targets
may also be involved since microRNAs can target thousands of
genes. Zeb1 is one of the crucial regulators of epithelial-to-
mesenchymal transition (EMT)  and has been shown to play a
major role in invasion and metastasis of epithelial tumors .
The relevance of ZEB proteins to tumor progression has been
studied in several human cancers. Expression of ZEB1 correlated
with an aggressive phenotype in various histological types of
endometrial carcinoma and was detected in the sarcomatous
compartment of endometrial carcinosarcoma . In colon
cancer, ZEB1 was expressed at the invasive front of tumors, in
association with the transient loss of basement membranes .
Reciprocal expression of ZEB1 and E-cadherin has also been
observed in non-small cell lung carcinoma . A direct
correlation between ZEB1 immunoreactivity and Gleason grade
has been reported in human prostate tumors  and in bladder
cancer, ZEB1 has been reported to be over-expressed and
responsible for enhanced motility . In the present study, we
found that over-expression of miR-23b resulted in suppression of
oncogene Zeb1 in the bladder cancer cells suggesting that miR-
23b can mediate EMT, thereby representing a possible mecha-
nism through which it affects bladder cancer migration and
In conclusion, this study shows that miR-23b has diagnostic/
prognostic significance and directly targets oncogenic Zeb1 in
bladder cancer. miR-23b over-expression resulted in suppression
of the bladder cancer cell proliferation and invasion, inducing
apoptosis, and cell cycle arrest. Finally, this study indicates that
miR-23b over-expression may be a therapeutically useful strategy
for the treatment of bladder cancer.
We thank Dr. Roger Erickson for his support and assistance with the
preparation of the manuscript.
Conceived and designed the experiments: SM AAD RD. Performed the
experiments: SM AAD GD. Analyzed the data: SM AAD GD SS IC KG
YT RD SY. Contributed reagents/materials/analysis tools: SM AAD GD
SS IC KG YT RD SY. Wrote the paper: SM. Data analysis: SM RD.
Edited the manuscript: SM RD.
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