Moreover, ectopic expres-sion of miR-203 promoted the apoptosis of human bladder cancer cell lines and inhibited cell proliferation, whereas its depletion increased cell growth.. We demo
Trang 1repressing bcl-w expression
Juanjie Bo, Guoliang Yang, Kailing Huo, Haifeng Jiang, Lianhua Zhang, Dongming Liu and
Yiran Huang
Department of Urology, Renji Hospital, Shanghai Jiaotong University, China
Introduction
Human bladder cancer is the fourth most common
malignancy in men, and the tenth most common in
women [1] Molecular and pathological studies suggest
that bladder cancers comprise at least two major
groups The majority of malignant bladder tumors are
urothelial cell carcinomas evolved from the epithelial
lining of the bladder wall [2] The urothelial
carcino-mas are noninvasive papillary tumors that commonly
recur, but rarely progress [3] Patients with tumors in
this group require long-term monitoring Invasive
bladder tumors are more aggressive, presenting with
penetration of the basement membrane or invasion
into muscle Patients with invasive disease have a
much worse prognosis, with a 5-year survival rate of
50% [4] The high frequency of recurrence of
noninva-sive urothelial carcinoma and the poor prognosis of
invasive bladder tumors highlight the necessity for
development of effective targeted therapies [5] How-ever, the lack of effective therapies for advanced blad-der cancer is related to poor unblad-derstanding of the molecular mechanisms underlying the progression of this disease [6]
Apoptosis is believed to be the major mechanism of chemotherapy⁄ radiotherapy-induced cell death in blad-der cancer [7] Unfortunately, many tumor cells evade drug-induced death signals, and certain oncogenes such
as bcl-2 and bcl-w can become overexpressed, resulting
in chemotherapy⁄ radiotherapy resistance [8] Bcl-2 family proteins are essential regulators of apoptosis, and bcl-w is anti-apoptotic member of this family Bcl-w maintains cell viability by preventing mitochon-dria-mediated apoptosis [9,10] Overexpression of bcl-w can prevent cell death, and is a potential mediator of resistance to several chemotherapeutic drugs [11]
Keywords
apoptosis; bcl-w; bladder cancer; miR-203;
proliferation
Correspondence
D Liu, Department of Urology, Renji
Hospital, School of Medicine Shanghai
Jiaotong University, No 145 Shan Dong
Middle Road, Shanghai 200001, China
Fax: +86 21 6373 0455
Tel: +86 21 6373 0455
E-mail: dmliu@hotmail.com
(Received 16 November 2010, revised 13
December 2010, accepted 22 December
2010)
doi:10.1111/j.1742-4658.2010.07997.x
It is increasingly clear that microRNAs (miRNAs) play an important role
in many diseases, including tumorigenesis However, the mechanisms by which miRNAs regulate bladder cancer development remain poorly under-stood Here, we evaluated the expression of microRNA-203 (miR-203) in bladder cancer tissues using real-time PCR, and defined the target genes and biologically functional effect using luciferase reporter assay, flow cytometry and western blot analysis We first verified that the expression of miR-203 was decreased in bladder cancer tissues Moreover, ectopic expres-sion of miR-203 promoted the apoptosis of human bladder cancer cell lines and inhibited cell proliferation, whereas its depletion increased cell growth
We further verified that miR-203 directly targeted 3¢-untranslated region of the bcl-w gene, and decreased its expression in vitro and in vivo Western blot analysis also showed that the expression level of miR-203 was nega-tively correlated with bcl-w level in tumor tissues These data suggest an important role for miR-203 in the molecular etiology of bladder cancer and implicate the potential application of miR-203 in bladder cancer therapy
Abbreviations
miRNA, microRNA; miR-203, microRNA-203; UTR, untranslated region.
Trang 2However, the regulation of endogenous bcl-w
expres-sion is complex For example, Michela et al showed
the role of Akt in apoptosis resistance by interacting
with bcl-w [12] They demonstrated that Akt interacted
with the N- and C-terminal sequences of the bcl-w
protein, and phosphorylated bcl-w both in vitro and in
the intact cell Lapham et al [13] demonstrated that
TCF4 and beta-catenin interacted with the bcl-w
pro-moter, and the activity of the bcl-w promoter was
increased or decreased, respectively, by overexpression
of beta-catenin or dominant negative TCF4 Lin et al
[14] showed that bcl-w is a direct target of miR-122
that functions as an endogenous apoptosis regulator in
these HCC-derived cell lines
There is increasing evidence that post-transcriptional
regulation of gene expression, mediated by
micro-RNAs (mimicro-RNAs), plays an important role in the
con-trol of cells proliferation, apoptosis and tumorigenesis
[15–17] miRNAs are noncoding RNAs that have been
highly conserved during evolution and have emerged
recently as potent regulators of gene expression
Over-expression of oncogenic miRNAs and underOver-expression
of tumor suppressor miRNAs play pivotal roles
in tumorigenesis Gottardo et al [18] compared the
miRNA profile of bladder cancers and noncancer
blad-der tissues, and found that 10 miRNAs were
upregu-lated MicroRNA-203 (miR-203), possessing a tumor
suppressive activity, was also downregulated in bladder
cancer tissues [19,20] However, the relationship
between miR-203 and bladder cancer is remains
unknown
In this study, we investigated the role of miR-203 in
bladder cancer development We demonstrated that
expression of miR-203 was downregulated in bladder
cancer tissues, and decreased expression of miR-203
contributed to cells proliferation Finally, we confirmed
that bcl-w is the direct target of miR-203
Results
Expression of miR-203 is significantly
downregulated in bladder cancer
In order to assess the role of miR-203 in bladder
can-cer development, we first evaluated the different
expression of miR-203 in bladder cancer tissues and
normal bladder tissues by using quantitative real-time
PCR Figure 1 shows that miR-203 was expressed at
low levels in bladder cancer tissue, and miR-203 levels
were increased in normal bladder tissue The mean
level of miR-203 expressed in normal bladder tissues
was 2.7, whereas the mean level of miR-203 expressed
in bladder cancer tissues was 1.4 (Fig 1) These data
indicate that down-regulation of miR-203 may be related to bladder cancer development
miR-203 promotes cells apoptosis in vitro
To study the physiological role of miR-203 in cell growth, bladder cancer cell lines treated with miR-203,
or microRNA control, were analyzed Figure 2A shows that overexpression of miR-203 promoted cell apoptosis in T24 cells, a bladder cancer cell line More-over, compared with the nonspecific microRNA con-trol (NC), miR-203 inhibited T24 cells proliferation (Fig 2B), whereas its depletion promoted cell growth (Fig 2C) These data suggest that miR-203 negatively regulates the growth of bladder cancer cells
bcl-w is a target gene of miR-203
In order to identify the target genes of miR-203 in regu-lating bladder cancer cell apoptosis, we searched for candidate genes using TargetScan 5.1 (http://www targetscan.org/) and miRBase (http://www.mirbase org/) microRNA databases The bcl-w gene is one of predicted target genes of miR-203 (Fig 3A), and is an important member of the bcl-2 family described as one
of the anti-apoptotic protein So, we constructed a
fire-fly luciferase reporter containing the 3¢-untranslated region (UTR) of bcl-w We then cotransfected HEK293 cells with the pGL3-promoter–bcl-w–3¢-UTR and miR-203 The reporter assay showed that miR-203 was
0.0
Normal Tumor
*
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 5.0
4.5
Fig 1 miR-203 was downregulated in bladder tumor tissues Analy-sis of miR-203 expression level was performed in bladder tumor tissues (n = 13) or normal bladder tissues (n = 9) Total RNA was subjected to real-time RT-PCR to analyze the expression level of miR-203 in each sample U6 was used as reference for miRNAs Each sample was analyzed in triplicate The 2)DDCtmethod was used
to determine the relative quantization of gene expression levels.
*P < 0.05.
Trang 3able to significantly repress luciferase expression
of pGL3-promoter–bcl-w–3¢-UTR (Fig 3B), and
mutation of four nucleotides in the miR-203 target
sequence led to complete abrogation of the suppressive
effect (Fig 3B) Moreover, we observed that ectopic
expression of miR-203 decreased the bcl-w protein
content in a time-dependent manner in T24 cells
(Fig 3C) These observations confirm that miR-203
inhibits endogenous bcl-w in bladder cancer cells
miR-203 expression is inversely related to bcl-w
expression in bladder cancer
To further examine the relationship between miR-203
and bcl-w in bladder cancer tissues, the expression of
miR-203 and bcl-w were detected Our data showed
that miR-203 levels were decreased in bladder cancer
samples bcl-w expression in bladder cancer samples
was analyzed by western bloting The bcl-w levels in
bladder cancer samples were significantly higher than
in normal tissue samples (Fig 4) Importantly, Fig 4 shows that bcl-w expression was increased in bladder cancer tissues that showed significantly decreased
miR-203 expression
miR-203 suppresses bladder cancer progression
by repressing bcl-w Bcl-w is an important determinant of cells prolifera-tion or apoptosis in cancer development, and ele-vated expression of the bcl-w resulted in abnormal cell growth with reduced capability for apoptosis in many cancer types Therefore, we thought that the role of miR-203 in bladder cancer cells growth was mediated by modulating bcl-w expression Figure 5 shows that T24 cell proliferation was restored when bcl-w was re-expressed in cells treated with miR-203 These data confirm that miR-203, at least in part, inhibited bladder cancer progression by repressing bcl-w
0
0
0
0.5
1
1.5
2
2.5
3
3.5
5
10
15
NC miR-203
*
*
*
*
*
NC
NC
miR-203
miR-203-inhibitor
12 0 0.1
0.2
0.3 0.4 0.5
0 0.1 0.2 0.3 0.4 0.5
0.6
0.6 0.7
miR-203
1.32 14.35 4.94
miR-203-inhibitor T24 cells no-specific inhibitor
NC
T24 cells
24 48 60 72
h
h
5
Annexin V-FITC
15
A
B
C
Fig 2 miR-203 inhibited bladder tumor cells proliferation (A) T24 cells were treated with mature miR-203 and apoptosis induc-ers, and apoptosis was detected using the flow cytometry Overexpression of miR-203 promoted cell apoptosis in T24 cells (B) T24 cells were transiently transfected with miR-203 mimics, and at the indicated time points, the numbers of cells per well were measured by the absorbance (450 nm) of reduced WST-8 (C) T24 cells were tran-siently treated with miR-203 inhibitor, and the number of cells per well was measured
by the absorbance (450 nm) of reduced 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-isulfophenyl)-2H-tetrazolium The results show data from at least three independent experiments, expressed as the mean ± SD *P < 0.05.
Trang 4miRNAs have been reported to be involved in several
important biological events, such as tumorigenesis, cell
differentiation, proliferation and apoptosis miRNAs
are also known to act as regulators in bladder cancer
cell growth, and have recently been reported to
regu-late bladder cancer progression [21,22] Some
miRNAs, including miR-145, miR-133a and miR-203,
were markedly downregulated in bladder cancer, and
miR-145 and miR-133a are involved in the regulation
of FSCN1 expression and possibly in the development
of bladder cancer [19,22] However, the relationship
between miR-203 and bladder cancer is still unknown
Here we found that miR-203 suppresses bladder cancer
development by repressing bcl-w expression
Bcl-2 family proteins regulate mitochondrial
apopto-sis downstream of diverse stressors Bcl-w is an
impor-tant anti-apoptotic member of the bcl-2 family
Depletion of bcl-w has been proven to sensitize cells to
apoptosis [23,24] Increased bcl-w protein levels are
commonly observed in various types of cancers,
including colonic, cervical and breast cancer cells [9]
In gastric adenocarcinomas, bcl-w suppresses cancer
cell death by blocking SAPK⁄ JNK activation [25]
Bcl-w expression is modulated by a Met⁄ HGF receptor (c-met) in human colorectal cancers Downregulation
of bcl-w by miR-122 resulted in a decrease in the Bcl-w⁄ Bax ratio, ultimately leading to apoptosis in HCC-derived cell lines [14] However, the regulation of bcl-w expression is complex, and its in-depth research con-tributes to cancer treatment
The function of miR-203 was recently reported Wijnhoven et al [26] showed that levels of miR-203
0 bcl-w Actin T1 T2 T3 T4
*
*
*
*
N1 N2 N3 N4 0.5
1 1.5 2 2.5
3 3.5 4 4.5 5
Fig 4 miR-203 expression is inversely related to bcl-w expression
in bladder tissues Bcl-w was decreased in normal bladder tissues that showed increased miR-203 expression, whereas bcl-w was increased in bladder tumor tissues that showed decreased miR-203 expression T, tumor tissues; N, normal tissues b-Actin was used
as reference for RNAs *P < 0.05.
0
h
miR-203 miR-203 + pcDNA bcl-w
* * NC
0.1 0.2 0.3
0.4 0.5 0.6 0.7
Fig 5 miR-203 suppresses bladder cancer progression by bcl-w modulation Bcl-w was re-expressed in T24 cells treated with miR-203, and the numbers of cells per well were measured by the absorbance (450 nm) of reduced 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-isulfophenyl)-2H-tetrazolium at 12, 24, 48, 60 and 72 h, respectively The results show data from at least three independent experiments, expressed as the mean ± SD *P < 0.05.
Bcl-w 3′-UTR
A
B
C
CCCAAUUUUUAAAUCCAUUUCAU
CCCAAUUUUUAAAUCCAAAAGAU
GAUCACCAGGAUUUGUAAAGUG 5′
5′
3′
Bcl-w 3′-UTR-mut
hsa-miR-203
0
NC miR-203
Hek293 cells
5
10
15
20
1.2 1 0.8 0.6 0.4 0.2 0 Wildtype Mutation
NC miR-203
bcl-w
Actin
bcl-w Actin
Fig 3 miR-203 inhibits bcl-w expression (A) Schematic
representa-tion of the miR-203 site in bcl-w 3¢-UTR (B) The 3¢-UTR reporter
assay was carried out in HEK293 cells that overexpressed miR-203.
PGL3-promoter–bcl-w–3¢-UTR–WT or PGL3-promoter–bcl-w–3¢-UTR–
MUT was cotransfected with pRL-TK using Lipofectamine 2000.
Luciferase assays were performed 24 h after transfection using the
Dual-Luciferase Reporter Assay System (Promega) Firefly luciferase
activity was standardized to Renilla luciferase control An asterisk
indicates significantly different from the NC (P < 0.05) (C) Western
blot analysis for endogenous bcl-w protein level using antibodies
against bcl-w b-Actin was used as reference for RNAs.
Trang 5were high in normal squamous epithelium and low in
columnar epithelia, and dysregulation of miR-203
could contribute to metaplastic and neoplastic
pro-cesses in the oesophageal mucosa Mathe et al [27]
demonstrated that miR-203 expression was reduced in
cancerous compared with noncancerous tissue, and
was potentially clinically useful for developing
prog-nostic biomarkers and identifying novel drug targets
and therapies However, the relationship between
miR-203 and bladder cancer is unknown Our results
showed that miR-203 levels were decreased in bladder
cancer tissues Overexpression of miR-203 promoted
bladder cancer cell apoptosis and inhibited cells
prolif-eration We identified that miR-203 targeted the bcl-w
gene The reporter assay showed that miR-203 was
able to significantly repress luciferase-contained bcl-w–
3¢-UTR expression Western blot analysis also showed
that miR-203 significantly inhibited the bcl-w protein
level in T24 cells
In conclusion, our data demonstrate that miR-203
suppresses bladder cancer cell proliferation by
target-ing bcl-w Thus, miR-203 is an important regulator in
the development of bladder cancer and implicates the
potential application of miR-203 in bladder cancer
therapy
Materials and methods
Tissue samples
Human bladder tissues were obtained with informed
con-sent from Renji Hospital affiliated to School of Medicine
Shanghai Jiaotong University Normal and pathologically
diagnosed biopsy specimens were obtained from patients
with bladder tumors who underwent radical cystectomy or
endoscopic resection for primary or recurrent TCC
Cell culture
Human bladder cancer cells T24 were obtained from the
American Type Culture Collection and were maintained in
RPMI 1640 with 10% fetal bovine serum (Gibco, New
York, USA), and were cultured at 37C with 5% CO2
The human embryonic kidney epithelial cell HEK-293 was
maintained in DMEM with 10% FBS and were cultured at
37C with 5% CO2
Real-time PCR
Total RNA was extracted using Trizol reagent (Invitrogen,
New York, USA), and the reverse-transcription reactions
were carried out using an M-MLV Reverse Transcriptase
kit (Invitrogen) The gene-specific stem–loop RT primers for
miRNAs released by the Sanger Institute were designed according to Chen et al [28] Real-time PCR was performed using a standard SYBR Green PCR kit (Toyobo, Osaka, Japan) protocol on Rotor-Gene RG-3000A (Corbett Life Science, Sidney, Australia) U6 and b-actin were used as ref-erences for miRNAs and RNAs, respectively Each sample was analyzed in triplicate The 2)DDCtmethod was used to determine the relative quantitation of gene expression levels
Luciferase reporter assay
For luciferase reporter assay, HEK293 cells (1–3· 104 cellsÆwell)1) were plated in a 24-well plate and then cotrans-fected with 20 mm of either miR-203 or microRNA control (NC), 40 ng of either pGL3-promoter–bcl-w–3¢-UTR–WT
or pGL3-promoter–bcl-w–3¢-UTR–MUT and 4 ng of
pRL-TK (Promega, Wisconsin, USA), using Lipofectamine
2000 HEK293 cells were collected 24 h after transfection and analyzed using the Dual-Luciferase Reporter Assay Sys-tem (Promega) The pRL-TK vector that provided the con-stitutive expression of Renilla luciferase was cotransfected as
an internal control to correct the differences in both trans-fection and harvest efficiencies Transtrans-fections were done in triplicate and repeated at least three times in independent experiments
Western blot analysis and antibodies
Western blot analysis to assess bcl-w and b-actin expression was performed as previously described [29] The anti-(bcl-w) primary Ig and anti-(b-actin) primary Ig were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA)
Flow cytometric analysis
T24 cells transfected with miR-203 (4· 105) were plated in six-well plates and apoptosis inducers A (Apopisa) and B (Apobid) (1 : 1000; Beyotime, Jiangsu, China) were added
to the culture After a 24-h incubation, the cultures were stained with annexin V-fluorescein isothiocyanate and apoptosis rates were analyzed using a flow cytometer (FACSCalibur, BD Biosciences, CA, USA)
Cell proliferation assay
Cell proliferation assays were performed with a Cell Count-ing Kit-8 (Dojindo, Kumamoto, Japan) [30] Cells were pla-ted in 24-well plates in triplicate at 1–2 · 105
cellsÆwell)1 and cultured in the growth medium Then cells were treated with mature miR-203 (miR-203 mimics, MIMAT0000264; Qiagen, Germany) or with miR-203 inhibitor
(anti-miR-203, MIN0000236; Qiagen, Hilden, Germany), the numbers
of cells per well were measured by the absorbance (450 nm)
of reduced
Trang 62-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-isulfophenyl)-2H-tetrazolium (monosodium salt) at the
indicated time points
Plasmid pcDNA–bcl-w was constructed by introducing a
BamHI–EcoRI fragment containing the bcl-w cDNA into
the same sites in pcDNA3.1
Statistical analysis
All data are expressed as mean ± SD from at least three
separate experiments The differences between groups were
analyzed using Student’s t-test The difference was deemed
statistically significant at P < 0.05
Acknowledgements
This work was supported by Shanghai Municipal
Nat-ural Science Foundation (09ZR1418000)
References
1 Lacey JR Jr, Devesa SS & Brinton LA (2002) Recent
trends in breast cancer incidence and mortality Environ
Mol Mutagen 39, 82–88
2 Egerod FL, Bartels A, Fristrup N, Borre M, Orntoft
TF, Oleksiewicz MB, Brunner N & Dyrskjot L (2009)
High frequency of tumor cells with nuclear Egr-1
pro-tein expression in human bladder cancer is associated
with disease progression BMC Cancer 9, 385
3 Seiler R, von Gunten M, Thalmann GN & Fleischmann
A (2010) Pelvic lymph nodes: distribution and nodal
tumour burden of urothelial bladder cancer J Clin
Pathol 63, 504–507
4 Kim WJ, Kim EJ, Kim SK, Kim YJ, Ha YS, Jeong P,
Kim MJ, Yun SJ, Lee KM, Moon SK et al (2010)
Predictive value of progression-related gene classifier in
primary non-muscle invasive bladder cancer Mol
Cancer 9, 3
5 Tomlinson DC, Lamont FR, Shnyder SD & Knowles
MA (2009) Fibroblast growth factor receptor 1
pro-motes proliferation and survival via activation of the
mitogen-activated protein kinase pathway in bladder
cancer Cancer Res 69, 4613–4620
6 Ismaili N, Heudel PE, Elkarak F, Kaikani W, Bajard
A, Ismaili M, Errihani H, Droz JP & Flechon A (2009)
Outcome of recurrent and metastatic small cell
carci-noma of the bladder BMC Urol 9, 4
7 Sanchez-Salas R, Duque Ruiz GI, Watson J, Rocha E,
Barret E, Cathelineau X, Rozet F, Galiano M &
Vallancien G (2009) Comprehensive surgical and
chemotherapy treatment for invasive bladder cancer
Acta Urol Esp 33, 1062–1068
8 Matsui Y, Hadaschik BA, Fazli L, Andersen RJ, Gleave
ME & So AI (2009) Intravesical combination treatment
with antisense oligonucleotides targeting heat shock
pro-tein-27 and HTI-286 as a novel strategy for high-grade bladder cancer Mol Cancer Ther 8, 2402–2411
9 O’Reilly LA, Print C, Hausmann G, Moriishi K, Cory
S, Huang DC & Strasser A (2001) Tissue expression and subcellular localization of the pro-survival molecule Bcl-w Cell Death Differ 8, 486–494
10 Crawford AC, Riggins RB, Shajahan AN, Zwart A & Clarke R (2010) Co-inhibition of BCL-W and BCL2 restores antiestrogen sensitivity through BECN1 and promotes an autophagy-associated necrosis PLoS ONE
5, e8604
11 Huang Z (2000) Bcl-2 family proteins as targets for anticancer drug design Oncogene 19, 6627–6631
12 Garofalo M, Quintavalle C, Zanca C, De Rienzo A, Romano G, Acunzo M, Puca L, Incoronato M, Croce
CM & Condorelli G (2008) Akt regulates drug-induced cell death through Bcl-w downregulation PLoS ONE 3, e4070
13 Lapham A, Adams JE, Paterson A, Lee M, Brimmell
M & Packham G (2009) The Bcl-w promoter is acti-vated by beta-catenin⁄ TCF4 in human colorectal carci-noma cells Gene 432, 112–117
14 Lin CJ, Gong HY, Tseng HC, Wang WL & Wu JL (2008) miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines Biochem Bio-phys Res Commun 375, 315–320
15 Esquela-Kerscher A & Slack FJ (2006) Oncomirs – microRNAs with a role in cancer Nat Rev Cancer 6, 259–269
16 Hwang HW & Mendell JT (2006) MicroRNAs in cell proliferation, cell death, and tumorigenesis Br J Cancer
94, 776–780
17 Saito Y, Suzuki H, Tsugawa H, Nakagawa I, Matsu-zaki J, Kanai Y & Hibi T (2009) Chromatin remodeling
at Alu repeats by epigenetic treatment activates silenced microRNA-512-5p with downregulation of Mcl-1 in human gastric cancer cells Oncogene 28, 2738–2744
18 Gottardo F, Liu CG, Ferracin M, Calin GA, Fassan
M, Bassi P, Sevignani C, Byrne D, Negrini M, Pagano
F et al (2007) Micro-RNA profiling in kidney and bladder cancers Urol Oncol 25, 387–392
19 Chiang Y, Song Y, Wang Z, Chen Y, Yue Z, Xu H, Xing C & Liu Z (2010) Aberrant Expression of
miR-203 and its clinical significance in gastric and colorectal cancers J Gastrointest Surg, doi:10.1007/s11605-010-1367-8
20 Castilla MA, Moreno-Bueno G, Romero-Perez L,
De Vijver KV, Biscuola M, Lopez-Garcia MA, Prat J, Matias-Guiu X, Cano A, Oliva E et al (2011) Micro-RNA signature of the epithelial–mesenchymal transition in endometrial carcinosarcoma J Pathol
223, 72–80
21 Huang L, Luo J, Cai Q, Pan Q, Zeng H, Guo Z, Dong W, Huang J & Lin T (2010) MicroRNA-125b
Trang 7suppresses the development of bladder cancer by
targeting E2F3 Int J Cancer, doi:10.1002/ijc.25509
22 Chiyomaru T, Enokida H, Tatarano S, Kawahara K,
Uchida Y, Nishiyama K, Fujimura L, Kikkawa N,
Seki N & Nakagawa M (2010) miR-145 and miR-133a
function as tumour suppressors and directly regulate
FSCN1 expression in bladder cancer Br J Cancer 102,
883–891
23 Ross AJ, Waymire KG, Moss JE, Parlow AF, Skinner
MK, Russell LD & MacGregor GR (1998) Testicular
degeneration in Bclw-deficient mice Nat Genet 18,
251–256
24 Print CG, Loveland KL, Gibson L, Meehan T,
Stylianou A, Wreford N, de Kretser D, Metcalf D,
Kontgen F, Adams JM et al (1998) Apoptosis
regula-tor bcl-w is essential for spermatogenesis but appears
otherwise redundant Proc Natl Acad Sci USA 95,
12424–12431
25 Lee HW, Lee SS, Lee SJ & Um HD (2003) Bcl-w is
expressed in a majority of infiltrative gastric
adenocarci-nomas and suppresses the cancer cell death by blocking
stress-activated protein kinase⁄ c-Jun NH2-terminal
kinase activation Cancer Res 63, 1093–1100
26 Wijnhoven BP, Hussey DJ, Watson DI, Tsykin A, Smith CM & Michael MZ (2010) MicroRNA profiling
of Barrett’s oesophagus and oesophageal adenocarci-noma Br J Surg 97, 853–861
27 Mathe EA, Nguyen GH, Bowman ED, Zhao Y, Budhu
A, Schetter AJ, Braun R, Reimers M, Kumamoto K, Hughes D et al (2009) MicroRNA expression in squa-mous cell carcinoma and adenocarcinoma of the esoph-agus: associations with survival Clin Cancer Res 15, 6192–6200
28 Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR Nucleic Acids Res
33, e179
29 Kapinas K, Kessler CB, Ricks T, Gronowicz G & Delany AM (2010) miR-29 modulates WNT signaling
in human osteoblasts through a positive feedback loop
J Biol Chem 285, 25221–25231
30 Qin W, Shi Y, Zhao B, Yao C, Jin L, Ma J & Jin Y (2010) miR-24 regulates apoptosis by targeting the open reading frame (ORF) region of FAF1 in cancer cells PLoS ONE 5, e9429