MicroRNA-1280 Inhibits Invasion and Metastasis by
Targeting ROCK1 in Bladder Cancer
Shahana Majid1, Altaf A. Dar2, Sharanjot Saini1, Varahram Shahryari1, Sumit Arora1, Mohd Saif Zaman1,
Inik Chang1, Soichiro Yamamura1, Takeshi Chiyomaru1, Shinichiro Fukuhara1, Yuichiro Tanaka1,
Guoren Deng1, Z Laura Tabatabai1, Rajvir Dahiya1*
1Department of Urology, VA Medical Center and UCSF, San Francisco, California, United States of America, 2Research Institute, California Pacific Medical Center, San
Francisco, California, United States of America
MicroRNAs (miRNAs) are non-protein-coding sequences that can function as oncogenes or tumor suppressor genes. This
study documents the tumor suppressor role of miR-1280 in bladder cancer. Quantitative real-time PCR and in situ
hybridization analyses showed that miR-1280 is significantly down-regulated in bladder cancer cell lines and tumors
compared to a non-malignant cell line or normal tissue samples. To decipher the functional significance of miR-1280 in
bladder cancer, we ectopically over-expressed miR-1280 in bladder cancer cell lines. Over-expression of miR-1280 had
antiproliferative effects and impaired colony formation of bladder cancer cell lines. FACS (fluorescence activated cell sorting)
analysis revealed that re-expression of miR-1280 in bladder cancer cells induced G2-M cell cycle arrest and apoptosis. Our
results demonstrate that miR-1280 inhibited migration and invasion of bladder cancer cell lines. miR-1280 also attenuated
ROCK1 and RhoC protein expression. Luciferase reporter assays demonstrated that oncogene ROCK1 is a direct target of
miR-1280 in bladder cancer. This study also indicates that miR-1280 may be of diagnostic and prognostic importance in
bladder cancer. For instance, ROC analysis showed that miR-1280 expression can distinguish between malignant and normal
bladder cancer cases and Kaplan-Meier analysis revealed that patients with miR-1280 high expression had higher overall
survival compared to those with low miR-1280 expression. In conclusion, this is the first study to document that miR-1280
functions as a tumor suppressor by targeting oncogene ROCK1 to invasion/migration and metastasis. Various compounds
are currently being used as ROCK1 inhibitors; therefore restoration of tumor suppressor miR-1280 might be therapeutically
useful either alone or in combination with these compounds in the treatment of bladder cancer.
Citation: Majid S, Dar AA, Saini S, Shahryari V, Arora S, et al. (2012) MicroRNA-1280 Inhibits Invasion and Metastasis by Targeting ROCK1 in Bladder Cancer. PLoS
ONE 7(10): e46743. doi:10.1371/journal.pone.0046743
Editor: Natasha Kyprianou, University of Kentucky College of Medicine, United States of America
Received July 24, 2012; Accepted August 30, 2012; Published October 4, 2012
Copyright: ? 2012 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 research was supported by the National Center for Research Resources of the National Institutes of Health through Grant Number RO1CA138642,
RO1CA130860, RO1CA160079 and 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: Rajvir Dahiya is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing
data and materials.
* E-mail: email@example.com
thought to regulate .90% of human genes . Deregulation of
miRNA expression has been identified in a number of cancers
[2,3], and accumulating evidence indicates that some miRNAs can
function as oncogenes or tumor suppressor genes. miRNAs are
expressed in a tissue-specific manner and can play important roles
in cell proliferation, apoptosis, and differentiation [4,5]. Inactiva-
tion of oncogenic miRNAs [6,7] or restoration of tumor-
suppressor miRNAs [8,9,10] may have great potential for cancer
Alterations in cellular functions such as cell proliferation,
adhesion and motility are based on the morphological changes
that result from actin cytoskeleton reorganization. Rho family
proteins interact with the actin cytoskeleton regulating formation
of stress fibers and focal adhesions within cells. Rho-associated
serine-threonine protein kinase, ROCK [11,12], one of the best
characterized downstream effectors of Rho, is activated when it
selectively binds to the active GTP-bound form of Rho. Activated
ROCK interacts with the actin cytoskeleton to promote stress-fiber
formation and assembly of focal contacts . Rearrangements of
the actin cytoskeleton are involved in cancer cell migration which
is central to the process of metastasis. Tang et al  examined the
effects of ROCK1 inhibition on the activity of upstream RhoA
and Rac1. ROCK1 indirectly diminishes the activity of upstream
RhoA by stimulating Tiam1-induced Rac1 activity. ROCK1
provides a feedback mechanism, mediating upstream Rac1 and
RhoA activity, thus reflecting the diverse effects of ROCK1 on the
functional balance of small GTPases . Rearranging the actin
cytoskeletal proteins in response to Rho is important for the ability
of tumor cells to metastasize . The Rho/ROCK pathway
plays role in cancer progression by regulating actin cytoskeleton
reorganization and a specific ROCK inhibitor was found to
suppress tumor growth and metastasis [16,17]. In prostate
carcinoma PC-3 cells, RhoA is a critical endogenous promoter
of cell invasion and migration . Inhibition of RhoA or its
major downstream effector, ROCK1, diminishes motility of
prostate carcinoma cells . In bladder cancer, the Rho/ROCK
PLOS ONE | www.plosone.org1October 2012 | Volume 7 | Issue 10 | e46743
pathway was reported to be involved in occurrence and
progression of bladder cancer . These observations suggest
that the Rho/ROCK pathway may be a molecular target for
prevention of cancer invasion and metastasis. Here for the first
time we report on the tumor suppressor activity of microRNA-
1280 (miR-1280) in bladder cancer and show that it bladder
cancer migration and invasion by directly targeting ROCK1.
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. 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 and J82 cells
were cultured in Minimum Essential Media (MEM) supplemented
with 10% FBS.
Plasmids, Precursors and Transfection
TaqMan probes and precursors for hsa-miR-1280 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. Lipofectamine 2000
(Invitrogen) was used for all transfections.
Figure 1. miR-1280 expression is downregulated in bladder cancer. A) Quantitative RT-PCR analysis of miR-1280 in cell lines. B) Fluorescence
In-situ hybridization (FISH) in cell lines. C) Quantitative real time PCR analysis of mir-23b expression in matched Laser-Captured Microdissected tissue
samples. (T/N- Tumor/Normal).
miR-1280 in Bladder Cancer
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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 the 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
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
In Situ Hybridization
In situ hybridization was performed as described previously .
Briefly cell lines were stained using DIG-labeled locked nucleic
acid (LNA)-based probes specific for mir-1280 following the
manufacturer’s protocol (Exiqon,Inc Woburn, MA) and detected
using anti-DIG-Fluorescein, Fab Fragments (Roche Applied
Science, Indianapolis, IN).
Figure 2. Diagnostic and prognostic significance of miR-1280 in bladder cancer. A) Clinicopathological characteristics of patient cohort. B)
ROC curve analysis showing performance of miR-1280 expression to discriminate between malignant and non-malignant tissue samples. C) Kaplan-
Meier analysis for overall survival based on miR-1280 expression.
miR-1280 in Bladder Cancer
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Cell Viability and Clonability Assay
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
protocol. Absorbance was measured at 490 nm using Spectra-
MAX 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 untill 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 resus-
pended 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 tissue
culture incubator. After 16 hours, non-migrated/non-invading
cells were removed from upper side of transwel 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 70–80% confluent 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
Figure 3. Transient transfection of miR-1280 inhibits bladder cancer cell proliferation and colony formation capability of bladder
cancer cells. A) Transient transfection of miR-1280 precursor significantly increased expression of miR-1280 in bladder cancer cells. B) Proliferation of
J82 and T24 cells after miR-1280 transfection was significantly reduced compared to cont-miR. C) miR-1280 over-expression significantly inhibits
colony forming ability of bladder cancer cells.
miR-1280 in Bladder Cancer
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antibodies and visualized using enhanced chemiluminescence
(Pierce Biotechnology, Rockford, IL).
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-1280 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
GAATCGTGTGGGAT 39 and Reverse 59 ctagatcccacacgattcca-
cagactagcggccgcgagct 39. For the mutant 39UTR, the primer
sequences were: Forward 59 CGCGGCCGCTAGTCTGTG-
GAATCGTTCATACT 39 and reverse 59 ctagagtatgaacgattcca-
cagactagcggccgcgagct 39. For lucifease assay, T24 and J82 cells
were cotransfected with hsa-miR-1280 and pmirGLO Dual-
Luciferase miRNA target expression vectors with wild-type or
mutant target sequence using Lipofectamine 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
potential of miR-1280 to discriminate between malignant and
non-malignant samples. For disease progression, Kaplan-Meier
(log-rank test) analysis was performed.
Figure 4. miR-1280 induces cell cycle arrest in bladder cancer cells. A-B) FACS analysis shows miR-1280 over-expression induces G2-M cell
cycle arrest in J82 and T24 cells with a corresponding decrease in S-phase cells. Values are shown from triplicate experiments 6SD.
miR-1280 in Bladder Cancer
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Expression of miR-1280 in Bladder Tumors and Cancer
Expression of miR-1280 was examined by real-time PCR in
bladder cancer cell lines J82, T24 and compared to non-
malignant cell line SV-HUC1. The results indicated that miR-
1280 was downregulated in cancer cell lines (Figure 1A). In-situ
hybridization also confirmed the presence of miR-1280 expres-
sion (green signal) in SV-HUC1 cells compared to cancer cell
lines (Figure 1B). To examine the biological significance of miR-
1280, its expression was analyzed in laser captured microdis-
sected (LCM) human bladder tumor tissue and compared to
normal matched control tissue. The expression of miR-1280 was
found to be significantly downregulated in all the tumor samples
compared to their matched normal samples (Figure 1C). These
results indicate a putative tumor suppressor role for miR-1280
in bladder cancer.
Diagnostic and Prognostic Significance of miR-1280 in
Clinical demographics of the patient cohort are summarized in
Figure 2A. Receiver operating curve (ROC) analyses were
performed to evaluate the ability of miR-1280 expression to
discriminate between normal and tumor cases using tissue
samples. An area under the ROC curve (AUC) of 0.886
(P,0.0001; 95% CI=0.775 to 0.998) (Figure 2B) was obtained
suggesting that miR-1280 expression can discriminate between
malignant and non-malignant samples and hence can be used as a
diagnostic marker for bladder cancer though additional samples
may strengthen these results. To determine whether miR-1280 has
any prognostic significance, we divided cases into low miR-1280
(expression T/N,0.8 fold) and high miR-1280 (expression T/
N.0.8 fold) groups and performed Kaplan-Meier survival
analysis. In Kaplan-Meier analysis, the high miR-1280 group
had significantly higher overall survival probability compared to
the low miR-1280 group (Logrank Test p,0.02) (Figure 2C).
Figure 5. Reconstitution of miR-1280 induces apoptosis in bladder cancer cell lines. A–B) miR-1280 over-expression induces apoptosis in
J82 and T24 cells with a concomitant decrease in the viable number of cells. Values shown are from triplicate experiments 6SD.
miR-1280 in Bladder Cancer
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These findings suggest that miR-1280 has the potential to be a
diagnostic and prognostic marker for bladder cancer.
MicroRNA-1280 Overexpression Suppresses Bladder
Cancer Cell Proliferation and Colony Formation
To determined the functional significance of miR-1280
overexpression in bladder cancer, we transfected bladder cancer
cell lines J82 and T24 with miR-1280 precursors. miR-1280 was
significantly overexpressed in J82 and T24 cell lines after transient
transfection with miR-1280 precursor compared to the cont-miR
precursor (Figure 3A). Ectopic expression of miR-1280 signifi-
cantly decreased cell proliferation as compared to cells expressing
cont-miR (Figure 3B). miR-1280 transfected cells also had low
colony formation ability as the number of foci in miR-1280
expressing cells were decreased when compared with cont-miR
transfected cells (Figure 3C). These results indicate anti-prolifer-
ative effect of miR-1280 in bladder cancer.
miR-1280 Triggers Cell Cycle Arrest and Induces
Apoptosis in Bladder Cancer Cells
FACS (fluorescence activated cell sorting) analysis revealed that
re-expression of miR-1280 lead to a significant increase in the
number of cells in the G2-M phase of the cell cycle (17% to 35%)
while the S-phase population decreased from 15% to 8% in J82
cells (Figure 4A). Similar results were observed in T24 cells with an
increase in G2-M population of cells (6% to 19%) and a decrease
in S-phase population (12% to 6%), suggesting that miR-1280
triggers a G2-M arrest in miR-1280 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 12%) in response to miR-1280 overexpression compared to
cont-miR with a corresponding 10% decrease in the viable cell
population in J82 cells (Figure 5A). In T24 cells, an increase (2% to
12%) in apoptotic cells was observed with miR-1280 overexpres-
Figure 6. Ectopic expression of miR-1280 inhibits bladder cancer cell migration. A) Migration assays of J82 and T24 cells transfected with
miR-1280. B) Representative pictures of migration assay.
miR-1280 in Bladder Cancer
PLOS ONE | www.plosone.org7October 2012 | Volume 7 | Issue 10 | e46743
sion compared to cont-miR (Figure 5B). These results indicate a
tumor suppressor role for miR-1280 in bladder cancer.
Anti-migration/Invasion Effects of miR-1280 in Bladder
Overexpression of miR-1280 had anti-migratory and anti-
invasive effects on bladder cancer cell lines. Less absorbance was
observed at 560 nm with miR-1280 transfected cells compared to
cont-miR in the migration assay (Figure 6) and miR-1280
overexpression also significantly reduced the invasiveness of
bladder cancer cells (Figure 7).
miR-1280 Directly Targets Oncogene ROCK1
ROCK1 has been reported to be an important molecule that
drives bladder cancer migration and invasion. Using an online
microRNA target database we found oncogene ROCK1 as the
putative target of miR-1280 with complementary 39UTR sites for
the seed sequence of miR-1280 (Figure 8A). We performed
Western analysis for ROCK1 expression in miR-1280 transfected
cells and found that miR-1280 attenuated the protein expression
of ROCK1 compared to the cont-miR (Figure 8B). We also found
a decrease in the protein levels of RohC, another oncogenic
protein that is upstream of ROCK1. To check whether a direct
interaction is involved between miR-1280 and its target oncogene
ROCK1, we performed luciferase reporter assays. We found that
co-transfection of miR-1280 along with the wild type 39UTR of
oncogene ROCK1 caused a significant decrease in luciferase units
compared to controls (Figure 8C). These results suggest that miR-
1280 targets oncogene ROCK1 directly.
Though little is known about microRNA-1280, one study has
shown that miR-1280 is expressed in colon and pancreatic cancers
based on expression analysis of 19 colorectal and 17 pancreatic
human cancer samples . Here we for the first time report that
miR-1280 plays a tumor suppressor and has diagnostic and
prognostic potential in bladder cancer. We also performed
Figure 7. Overexpression of miR-1280 inhibits bladder cancer cell invasion. A) Invasion assay shows a significant decrease in the number of
invading J82 and T24 cells transfected with miR-1280. B) Representative pictures of invasion assay.
miR-1280 in Bladder Cancer
PLOS ONE | www.plosone.org8 October 2012 | Volume 7 | Issue 10 | e46743
functional analyses to confirm the anti-tumor effects of miR-1280
and show that miR-1280 directly targets oncogene ROCK1, an
important molecule in bladder cancer cell migration and invasion.
We also observed that miR-1280 is significantly downregulated
in bladder cancer cell lines and tumor tissues compared to non-
malignant cell line or normal tissues indicating that miR-1280
might be 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 [5,6]. 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-1280
was predictive of overall survival such that patients with higher
miR-1280 expression had longer overall survival compared to
patients with low miR-1280 expression. MicroRNA-1280 expres-
sion also distinguished malignant from normal tissues indicating
the diagnostic significance of miR-1280 in bladder cancer
although this needs to be confirmed in a larger cohort of tissue
samples. Our functional assays revealed that miR-1280 has anti-
proliferative effects, inducing cell cycle arrest and apoptosis in
bladder cancer. It also showed anti-migratory and anti-invasive
effect on bladder cancer cells.
Since ROCK1 is an important molecule that is involved in
bladder cancer migration and invasion , we examined whether
ROCK1 is a target of miR-1280 in bladder cancer. Overexpres-
sion of ROCK1 has been reported to occur in various cancers
[14,19] and the Rho/ROCK pathway has been found to be
associated with progression of bladder cancer . ROCK
mediates responses in the pathway initiated by Rho, and regulates
the reorganization of cytosleletal proteins such as formation of
stress fibers and focal adhesions . Rearranging cytoskeletal
proteins is important for the ability of tumor cells to metastasize
 and our study showed that miR-1280 attenuated expression
of oncogene ROCK1. Luciferase assays also revealed direct
interaction of miR-1280 and ROCK1. Therefore these results
indicate that miR-1280 inhibits migration/invasion and thus
Figure 8. miR-1280 directly targets oncogene ROCK1. A) Complimentary miR-1280 binding sequences in the ROCK1 39UTR. B) Western blot
analysis shows that miR-1280 represses translation of oncogenes ROCK1 and RhoC. C) Luciferase assays showing decreased reporter activity after co-
transfection of either the wild type or mutant Src-39UTR with miR-1280 in J82 and T24 cells. Mut- Mutated 39UTRROCK1 sequence. D) Schematic
representation of role of miR-1280 in bladder cancer.
miR-1280 in Bladder Cancer
PLOS ONE | www.plosone.org9 October 2012 | Volume 7 | Issue 10 | e46743
metastasis of bladder cancer that is mediated through downreg- Download full-text
ulation of oncogene ROCK1 (Figure 8D). We also found
decreased expression of oncogene RhoC that is upstream of
ROCK1. A previous study has reported that ROCK1 diminishes
activity of its upstream RhoA family members indirectly . By
the same principal, inhibition of ROCK1 by miR-1280 may have
an indirect inhibitory action on the RhoC oncogene.
In conclusion, our study is the first report to document the
tumor suppressor role of miR-1280 in bladder cancer. miR-1280
directly targets oncogene ROCK1, inhibiting migration/invasion
which are central to the process of metastasis. Various compounds
such as Y-27632, have been found to inhibit ROCK1. Our
findings indicate that restoration of tumor suppressor miR-1280
might be useful therapeutically either alone or in combination with
these compounds in 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 SS VS GD. Analyzed the data: SM AAD RD SS.
Contributed reagents/materials/analysis tools: SM AAD SS VS SA MSZ
IC SY TC SF YT GD. Wrote the paper: SM. Designed the research plan:
SM RD. Conducted experiments: SM AAD VS. Analyzed data: SM AAD
SS VS SA MSZ IC SY TC SF YT GD RD. Provided necessary reagents
for research: SM AAD SS VS SA MSZ IC SY TC SF YT GD. Wrote
manuscript: SM. Supervise the research and review manuscript: SM RD.
Board certified pathologist to identify tumor and normal tissues: ZLT.
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