miR-429 expression in bladder cancer and its correlation with tumor behavior and clinical outcome

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Kaohsiung Journal of Medical Sciences (2018) xx, 1e6

Available online at www.sciencedirect.com

ScienceDirect journal homepage: http://www.kjms-online.com

Original Article

miR-429 expression in bladder cancer and its correlation with tumor behavior and clinical outcome Chia-Lun Wu a,b, Jar-Yi Ho b,c, Shun-Hsing Hung d, Dah-Shyong Yu a,e,* a

Division of Urology, Department of Surgery, Tri-Service General Hospital, Taipei, Taiwan Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan c Graduate Institute of Pathology and Parasitology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan d Division of Urology, Department of Surgery, Chi-Mei Medical Center, Tainan, Taiwan e Department of Surgery, National Defense Medical Center, Taipei, Taiwan b

Received 4 July 2017; accepted 3 January 2018

KEYWORDS Bladder cancer; Cancer survival rate; microRNA-429; Tumor behavior

Abstract We previously showed that microRNA-429 (miR-429) played an important role in epithelialemesenchymal transition (EMT) of urothelial cell carcinoma of the bladder. We herein evaluated the expression of miR-429 in bladder cancer and its potential relevance to clinicopathological characteristics and patient survival. Relative expression levels of miR-429 in surgical bladder cancer tissue specimens obtained from 76 patients with bladder cancer were measured by chromogenic in situ hybridization. miR-429 expression was significantly higher in specimens from alive patients than expired patients in both of 5-year overall survival (OS) (0.59
0.09 vs. 0.27
0.12; p < 0.05) and 5-year recurrence-free survival (RFS) (0.63
0.10 vs. 0.33
0.10; p < 0.05). The univariate Cox proportional hazards analysis revealed that tumor grade, stage, and miR-429 expression were significantly associated with patient survival. In multivariate analysis, tumor stage and miR-429 expression were significantly associated with 5-year OS (hazard ratio [HR] 4.70, p < 0.001) and 5-year-RFS (HR 2.20, p < 0.05). The KaplaneMeier analysis showed that patients with miR-429 expression had significantly better 5-year OS and 5-year RFS rates than those without miR-429 expression (84.4% vs. 61.4%, p < 0.05 and 71.9% vs. 45.5%, p < 0.05, respectively). miR-429 may be considered as an adjunctive prognostic marker in addition to tumor grade and stage in bladder cancer. Copyright ª 2018, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. Division of Urology, Department of Surgery, Tri-Service General Hospital, No. 325, Section 2 Cheng-Gung Road, Neihu, 114, Taipei, Taiwan, ROC. E-mail address: [email protected] (D.-S. Yu). https://doi.org/10.1016/j.kjms.2018.01.001 1607-551X/Copyright ª 2018, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Wu C-L, et al., miR-429 expression in bladder cancer and its correlation with tumor behavior and clinical outcome, Kaohsiung Journal of Medical Sciences (2018), https://doi.org/10.1016/j.kjms.2018.01.001

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Introduction Bladder cancer, which ranks ninth in incidence among all cancers globally (6.1%), was the 13th deadliest cancer (2.3%) in 2012 [1]. In Taiwan, urothelial cell carcinoma (UCC) of bladder ranks 11th (2.1%) in incidence and was the 13th cause of cancer-related deaths (1.8%) in 2012 [2]. Current prognostic strategies, such as tumor grade, stage, size, and number of foci, have restricted utility for clinicians, given that they do not specifically reflect the clinical outcomes of bladder cancer patients [3]. Diagnosis and monitoring strategies for bladder cancer have been based on the integration of cystoscopy and urinary cytology [4]. MicroRNA (miRNA) is a small non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression. Many miRNAs were shown to have links with cancer and are accordingly referred to as oncomirs. Therefore, amidst the search for novel biomarkers, an important focus has been on the role of miRNAs in the development and prognosis of bladder cancer [5,6]. We previously demonstrated that miR-429 affected the biological behavior of bladder cancer cells through the reduction of zinc finger E-box-binding homeobox 1 (ZEB1) and b-catenin expression, thereby restoring E-cadherin expression and inactivating matrix metalloproteinase-2 (MMP-2) in UCC cell lines. These findings implicate miR429 as a potential progression marker in bladder cancer [7]. We conducted a retrospective study to further elucidate the potential role of miR-429 as a predictor of disease progression and outcome in patients with bladder cancer by correlation analyses.

Materials and methods Patients and tissue samples The study population included surgical bladder cancer specimens from 76 patients who were treated at TriService General Hospital (TSGH) in Taiwan between 2002 and 2006, in accordance of TSGH institutional review board guidelines (approval no: TSGH-IRB-099-05-262). At least five years of postoperative follow-up data were available for all patients. All excisional biopsies or therapeutic surgical specimens were diagnosed histologically. Clinical and histological information were obtained from patient charts and pathological reports. Tumor staging was recorded according to the AJCC Cancer Staging Manual (5th ed.) tumor-node-metastasis (TNM) staging system. Pathological grading of the bladder cancer was determined according to the World Health Organization histological grading criteria. Accurate survival data were provided by the Cancer Registry Group of TSGH.

C.-L. Wu et al. 3-min rinse in each of the 95% ethanol, 75% ethanol, and 50% ethanol solutions; one 5-min rinse in running tap water; 5min staining with Mayer’s hematoxylin solution. Slides were then rinsed once in running cold tap water for 5 min, ten times in 95% acid ethanol for 3 s each, and stained in eosin Y solution for 30 s. Slides were dehydrated once for 3 min in each of the 50% ethanol, 75% ethanol, and 95% ethanol solutions; and three times for 3 min in 100% ethanol. Next, slides were rinsed three times for 3 min in xylene for clearing. Slides were covered with glass slips and coded for blinded evaluation before evaluation using an Olympus BX41 microscope (Olympus, Tokyo, JP).

Chromogenic in situ hybridization Detection of miR-429 expression in bladder cancer tissue was achieved with the digoxin (DIG)-labeled specific probe 50 -ACGGTTTTACCAGACAGTATTA-30 (Phalanx Biotech Group, Hsinchu, Taiwan) using chromogenic in situ hybridization [8]. Tissue slides were deparaffinized and processed as follows: incubation at 75  C in an oven for 10 min, three 3min rinses in xylene; three 3-min rinses in 100% ethanol; one 3-min rinse in each of the 95% ethanol, 75% ethanol, and 50% ethanol solutions; and one 3-min rinse in running cold tap water. Afterwards, endogenous peroxidase activity and proteins were removed as follows: tissue slides were rinsed in 3% H2O2 for 15 min, H2O for 3 min, proteinase K (15 mg/mL, Sigma, USA) at 55  C for 60 min, H2O for 3 min, and 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min. Tissue slides were hybridized with the DIG-labeled miR-429 probe and incubated at 55  C for 16 h. The slides were washed with decreasing concentrations of saline-sodium citrate (SSC) buffer as follows: once for 10 min with 5  SSC at 55  C, twice for 10 min with 1  SSC at 55  C, twice for 30 min with 0.2  SSC at 55  C, once for 30 min with 0.2  SSC at room temperature, once for 10 min with PBS. Blocking and antibody hybridization were processed as follows: incubation for 15 min with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST), three washes with TBST, incubation with the mouse anti-DIG IgG antibody (Jackson Laboratory, Maine, USA) for 2 h, three washes with TBST, incubation with the goat polyclonal horseradish peroxidase-conjugated anti-mouse IgG antibody (Boster Biological Technology, CA, USA) for 1 h, three washes with TBST, incubation with 30 mg/mL 3,30 -Diaminobenzidine chromogen (DAKO, Glostrup, Denmark) for 5 min, one rinse with H2O for 5 min, and incubation in hematoxylin counterstain for 1 min. Slides were then covered with glass slips, coded, and subsequently evaluated under a standard light microscope at 40 and 400 magnifications. Staining intensity was graded as 0, 1, 2, or 3 for no staining, weak staining, moderate staining, and strong staining, respectively, as described before, by two observers, and results were recorded [9].

Hematoxylin & eosin staining Statistical analysis Three micrometer-thick sections were prepared from paraffin-embedded tissue blocks and stained by hematoxylin & eosin. Briefly, slides were deparaffinized and processed as follows: incubation at 75  C in an oven for 10 min; three 3min rinses in xylene; three 3-min rinses in 100% ethanol; one

Five-year overall survival was defined as the time interval between the year of surgery and death, and non-bladder cancer-related causes of death until the end of the observation period were censored. Five-year recurrence-free

Please cite this article in press as: Wu C-L, et al., miR-429 expression in bladder cancer and its correlation with tumor behavior and clinical outcome, Kaohsiung Journal of Medical Sciences (2018), https://doi.org/10.1016/j.kjms.2018.01.001

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miR-429 expression in bladder cancer

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survival was defines as the time interval between the year of primary surgery to the first evidence of local or distant recurrent disease. Survival rates were evaluated using KaplaneMeier curves with the log-rank test and the Cox proportional hazards regression model. P values < 0.05 were considered significant for all statistical analyses. Table 1 Correlation of miR-429 expression with patients’ dermatographics and pathological features. Total miR-429 expression (n Z 76) Negative (%) Positive (%)

60 y/o 63

7 (53.8) 37 (58.7)

Sex Female

19

12 (63.2)

Male

57

32 (56.1)

Histologic grade Low 25 High

10 (40.0)

51

34 (66.7)

TNM stage I 44

24 (54.5)

II

16

10 (62.5)

III

13

8 (61.5)

IV

3

2 (66.7)

a

Based on c2 test.

Demographic characteristics of patients Epidemiological and clinical information of the study cohort are summarized in Table 1. Majority of the patients were aged >60 years (63/76, 82.9%) and male (57/76, 75%). There were 44, 16, 13, and 3 clinical stage I, II, III, and IV bladder cancer cases, respectively.

pa

Association of miR-429 expression with tumor grade and stage

Staining grade (1 2 3) Age <60 y/o 13

Results

6 (46.2) (5 þ 1 þ 0) 26 (41.3) (18 þ 7 þ 1)

0.745

7 (36.8) (6 þ 1 þ 0) 25 (43.9) (17 þ 7 þ 1)

0.592

15 (60.0) (10 þ 5 þ 0) 17 (33.3) (13 þ 3 þ 1)

0.027

20 (45.5) (15 þ 0 þ 0) 6 (37.5) (5 þ 0 þ 1) 5 (37.5) (2 þ 3 þ 0) 1 (33.3) (1 þ 0 þ 0)

0.488

The positive staining rate of miR-429 was higher in lowgrade than in high-grade bladder cancer tissue specimens (60.0%, n Z 25 vs. 33.3%, n Z 51; p Z 0.027; Table 1, Fig. 1). The staining intensity of miR-429 was also higher in low-grade than in high-grade bladder cancer tissue specimens (0.80
0.15 vs. 0.40
0.09; p Z 0.020). There was no significant difference in miR-429 expression among different tumor stages, although the rate of miR-429 expression appeared to be higher in low-stage (stage I) than in high-stage (stage IIeIV) tumor specimens (p Z 0.488, Table 1).

Association of miR-429 expression with patient survival Univariate analysis with Cox proportional hazards regression model was performed including several parameters associated with patient survival including histologic grade, stage, and miR-429 expression. Negative miR-429 expression was significantly associated with worse 5-year overall survival and 5-year recurrence-free survival (Table 2). Higher positive staining rate of miR-429 with the 5-year overall and 5-year recurrence-free survival status of bladder cancer patients were observed in alive bladder cancer patients than expired bladder cancer patients (84.4% vs. 61.4%; p Z 0.046 and 71.9% vs. 45.5%;

Figure 1. Hematoxylin & eosin staining and histological scoring of miR-429 intensity. Hematoxylin & eosin staining of (a) lowgrade and (b) high-grade bladder tumor specimens and representative chromogenic in situ hybridization for miR-429 of (c) lowgrade and (d) high-grade bladder tumor specimens (400). (e) Correlation of miR-429 intensity with tumor grade in bladder cancer patients.

Please cite this article in press as: Wu C-L, et al., miR-429 expression in bladder cancer and its correlation with tumor behavior and clinical outcome, Kaohsiung Journal of Medical Sciences (2018), https://doi.org/10.1016/j.kjms.2018.01.001

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<0.001 <0.001 <0.001 <0.001

Abbreviations: HR, Hazard ratio; CI, confidence interval. a Based on the Cox regression model, with statistical significance (p < 0.05). b Categorized as positive and negative was set in miR-429 intensity 1 (positive) and miR-429 intensity <1 (negative) individually.

0.023

1 (ref) 2.766 (1.019e7.503) 1 (ref) 5.375 (1.255e23.014) 1 (ref) 12.535 (3.693e42.550) 32 44 25 51 44 32 miR429-positive miR429-negative b Histologic grade-low Histologic grade-high TNM stage-I TNM Stage IeIIeIV

N

0.503

1 (ref) 2.278 (1.058e4.904) 1 (ref) 3.147 (1.212e8.170) 1 (ref) 5.081 (2.395e10.781) 0.063 0.032

HR (95% CI)

1 (ref) 2.590 (0.950e7.061) 1 (ref) 1.678 (0.369e7.633) 1 (ref) 10.842 (3.049e38.555)

p b

HR (95% CI)

p

Multivariate Univariate

0.019

0.035

1 (ref) 2.205 (1.010e4.813) 1 (ref) 1.484 (0.542e4.060) 1 (ref) 4.701 (2.139e10.332)

p HR (95% CI) HR (95% CI)

p

Multivariatea Univariatea

5-year recurrence-free survival

a a

5-year overall survival

Correlation analysis of bladder cancer patients’ survival with miR-429 and various prognostic factors. Table 2

0.442

C.-L. Wu et al.

0.047

4

p Z 0.035), respectively. Furthermore, higher tumor stage and higher pathological grade were also associated with worse prognosis. However, diagnostic age and patient gender were not related to 5-year overall survival and 5year recurrence-free survival. Assessment of the association of miR-429 expression intensity with the 5-year overall survival and 5-year recurrence-free survival status revealed that miR-429 expression levels were higher in alive patients than expired patients (p < 0.05, Fig. 2a and b). To evaluate the prognostic value of miR-429, survival analyses were performed using the KaplaneMeier method. Negative miR-429 expression was significantly associated with worse overall survival and recurrence-free survival in bladder cancer (Fig. 3a and b; p Z 0.036 and 0.029, respectively). In multivariate analysis as shown in Table 2, negative miR-429 expression and clinical stage were independent risk factors for worse prognosis in bladder cancer. Negative miR-429 expression in bladder cancer specimens was associated with a 2.2-fold higher risk of worse prognosis than positive miR-429, including 5-year overall survival and 5year recurrence-free survival rates. Conversely, higher tumor stage was associated with a 10.8-fold higher risk for bladder cancer-associated death and 4.7-fold higher risk for worse prognosis in the study cohort.

Discussions miRNAs are recognized for their potential as biological markers for early diagnosis and prognosis as well as therapeutic targets for a variety of oncological diseases [4,10]. Exciting recent work suggests that the miR-200 family plays central roles in mediating the effects of epithelialemesenchymal transition (EMT) regulators in normal and malignant epithelial cells [11,12]. EMT is an essential mechanism activated during cancer invasion and metastasis and is characterized by loss of cell adhesion, inhibition of E-cadherin expression, and increased cell mobility [13]. miR-9, miR-182, miR-200b, and miR-200c were found to be related to tumor aggressiveness and prognosis in bladder cancer [5,6]. Simultaneously, loss of miR-200 expression was shown to promote EMT and resistance to epidermal growth factor receptor-based therapies [14]. miR-96 and miR-183 expression levels in the urine of bladder cancer patients were significantly higher than in that of healthy individuals. miR-96 and miR183 overexpression were correlated with tumor grade and pathological stage [15]. Ratert et al. observed a correlation between miR-141 and miR-205 expression levels and overall survival in bladder cancer patients [16]. Whereas the clinicopathological role of miR-429 has not been studied in bladder cancer, miR-429downregulation was reported in several cancer types including gastric cancer [17], breast cancer [18], renal cell carcinoma [19], colorectal carcinoma [20], and nasopharyngeal carcinoma [21]. However, contrary results were also reported, including miR-429 upregulation in certain cancer types [22e24]. For example, a previous study demonstrated that miR-429 inhibited migration and invasion in breast cancer [25]. miR-429 was also shown to inhibit the invasive ability in

Please cite this article in press as: Wu C-L, et al., miR-429 expression in bladder cancer and its correlation with tumor behavior and clinical outcome, Kaohsiung Journal of Medical Sciences (2018), https://doi.org/10.1016/j.kjms.2018.01.001

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Figure 2. Correlation of miR-429 intensity and survival rate. Correlation of miR-429 intensity with (a) 5-year overall survival and (b) 5-year recurrence free survival in bladder cancer patients.

Figure 3. KaplaneMeier curves for miR-429 expression. The KaplaneMeier curves showing miR-429 expression in association with (a) 5-year overall survival rate and (b) 5-year recurrence-free survival rate in bladder cancer patients.

gastric cancer by downregulating ZEB proteins, downstream targets of miR-429 [26]. In contrast, one study showed that miR-429 increased the metastatic capability of hepatocellular carcinoma by the Wnt pathway but not through EMT [27]. We previously found that miR-429 was closely related to tumor behavior in a cultured UCC cell line, including cellular differentiation, migration, and invasion through reversal of EMT by restoring E-cadherin [7]. Therefore, the current study was conducted for further confirmation of the potential ability of miR-429 as a predictor of disease progression and outcome in patients with bladder cancer. First, the current study revealed that miR-429 expression varied among different bladder cancer grades. miR-429 expression rate was lower in high-grade tumors than lowgrade tumors, indicating that miR-429 could be utilized as a predictor of tumor invasiveness and behavior. Decreased miR-429 expression in UCC cells can enhance tumor migratory ability, which usually underlies subsequent disease progression and recurrence. In our previous study, enhancing miR-429 expression led to a reduction in ZEB1 and b-catenin production, thereby restoring E-cadherin expression and subsequent inactivation of MMP-2 in UCC cells [7]. In the current study, there were no significant associations between miR-429 expression levels and age at diagnosis, patient gender, or stage of bladder cancer. Univariate analysis revealed that tumor grade, stage, and miR-429 expression level were significantly associated

with the 5-year overall survival and 5-year recurrence-free survival. Multivariate analysis demonstrated tumor stage and miR-429 expression level as two significant factors related to patient survival, including 5-year overall survival and 5-year recurrence-free survival. These findings reflect the negative impact of miR-429 on the invasiveness of UCC cells. These results are similar to other miRNAs associated with tumor grade, stage, and survival in bladder cancer patients [13e16]. Combination of these miRNAs as prognostic factors might significantly improve their predictive power for disease progression and outcomes in addition to their utility in tumor grading and staging. As shown in Table 1, the discrepancy between tumor grade and stage in relation to the expression of miR-429 in the current study might be due to the relatively small sample size of patients. Nevertheless, patients with muscle-invasive bladder cancer had worse survival than those with superficial cancer, as shown in Table 2. The inconsistency between univariate and multivariate analyses for survival outcomes related to tumor grade, stage, and miR-429 expression requires further verification in future studies, as the sample size of the current study was relatively small. In summary, the results of the current study revealed that in addition to tumor stage, miR-429 might be a potential clinical prognostic marker in bladder cancer patients.

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Research involving human participants and/or animals All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. For this type of study formal consent is not required. This article does not contain any studies with animals performed by any of the authors.

C.-L. Wu et al.

[11]

[12]

[13]

Informed consent [14]

Informed consent was obtained from all individual participants included in the study.

[15]

Acknowledgements The authors thank the Cancer Registry Group of TSGH for the clinical data providing and analysis. This study was funded by grants from the Ministry of Science and Technology, Republic of China (MOST 104-2314-B-016-040-MY3), Tri-Service General Hospital (TSGH-C103-063), National Defense Medical Center (MAB-104-075), and Chi Mei Medical Center (CMNDMC-103011).

[16]

[17]

[18]

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Please cite this article in press as: Wu C-L, et al., miR-429 expression in bladder cancer and its correlation with tumor behavior and clinical outcome, Kaohsiung Journal of Medical Sciences (2018), https://doi.org/10.1016/j.kjms.2018.01.001