Inhibiting cell migration and cell invasion by silencing the transcription factor ETS-1 in human bladder cancer

As one of the members of the ETS gene family, the transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS-1) plays key role in the regulation of physiological processes in normal cells and tumors. In this study, we aimed to investigate the relationship between the transcription factor ETS-1 and malignant phenotypes of bladder cancer. We demonstrated that ETS-1 was up-regulated in human bladder cancer tissue compared to paired normal bladder tissue. In order to evaluate the functional role of ETS-1 in human bladder cancer, vectors expressing ETS-1 shRNA and ETS-1 protein were constructed in vitro and transfected into the human bladder cancer T24 and 5637 cells. Our results showed that the transcription factor ETS-1 could promote cell migration and cell invasion in human bladder cancer, without affecting cell proliferation and apoptosis. In conclusion, ETS-1 plays oncogenic roles through inducing cell migration and invasion in human bladder cancer, and it can be used as a therapeutic target for treating human bladder cancer.


INTRODUCTION
Human bladder cancer is one of the most common cancers worldwide [1]. Although methods for early diagnosis of bladder cancer and more suitable surgeries were emerged, the mortality rate has not been decreased effectively [2]. One of these important reasons is that the potential mechanisms causing cancer progress are still poorly understood. Therefore, searching novel reliable biomarkers and revealing the molecular mechanism of bladder cancer for developing effective therapy are necessary.
As the first found member of the ETS transcription factors family, the transcription factor v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS-1) plays diverse roles in physiological and pathological processes, such as embryonic development, angiogenesis, and hematopoietic differentiation [3,4]. Furthermore, deregulated ETS-1 also has been observed in the carcinogenesis, invasion, and progression of various tumors [5,6,7]. One of the potential mechanisms of ETS-1is to activate the transcription of several proteases, such as matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator [5,6,7]. However, the functional character of ETS-1 in human bladder cancer is still unclear. Therefore, we aimed to study the expression pattern of ETS-1 and to determine its functional role in human bladder cancer.

ETS-1expression level was increased in bladder cancer
The relative expression level of ETS-1 was examined using real-time qPCR in a total of 42 patients with bladder cancer. The ETS-1 expression fold-change (bladder cancer tissue/matched normal tissue) in most patients were greater than zero in Figure 1A. Similarly, as shown in Figure 1B, The expression levels of ETS-1 were significantly upregulated in bladder cancer tissues compared with matched normal tissues. These results manifested that ETS-1 should play oncogenic role in bladder cancer. After that, we assessed the relationship between ETS-1 expression level and clinical-pathologic characteristics in 42 patients with bladder cancer. In Table 1, the results indicated that ETS-1 expression level was associated with the depth of invasion in these patients with bladder cancer, but it has no relationship with the age, the gender and the grade. These results suggested that ETS-1overexpression may be related to bladder cancer invasion.

Inhibition or activation of the expression of ETS-1 in bladder cancer
Bladder cancer 5637, T24 and UMUC-3 cells were cultured and treated with ETS-1 shRNA or the overexpression vector. The related expression level of ETS-1 was tested by real-time qPCR at 48 hours after treatment. The data indicated that the related expression level of ETS-1in 5637 (Figure 2A), T24 cells ( Figure 2B) and UMUC-3 ( Figure 2C) was observably downregulated by the specific ETS-1 shRNA. Also, the over-expression vector increased the expression level of ETS-1in 5637 ( Figure 2D), T24 ( Figure 2E) and UMUC-3 ( Figure 2F) cells.

ETS-1 promoted bladder cancer cell migration in vitro
In order to evaluate the role of ETS-1 in regulating human bladder cancer migration, bladder cancer 5637, T24 and UMUC-3 cells were treated with ETS-1 shRNA or the over-expression vector and analyzed by the cell migration assay afterwards. Compared to the negative control group, cell migration ability was significantly inhibited by the ETS-1 shRNA in 5637 cells ( Figure 3A), T24 cells ( Figure 3B) and UMUC-3 cells ( Figure 3C). In contrast, cell migration ability was significantly enhanced by the ETS-1over-expression vector in 5637 cells ( Figure 3D), T24 cells ( Figure 3E) and UMUC-3 cells ( Figure 3F). These results demonstrated that ETS-1 positively facilitate the bladder cancer cell migration.

ETS-1 promoted bladder cancer cell invasion in vitro
We further investigated whether ETS-1also promotes cell invasion in bladder cancer. Here, the invasion assay was used to verify our hypothesis. As Shown in Figure 4A-4C, compared with the negative control group, the cell invasion ability was extremely suppressed in 5637 cells, T24 cells and UMUC-3 cells which were transfected with ETS-1 shRNA. As shown in Figure 4D-4F, compared with the negative control group, the cell invasion ability was extremely enhanced in 5637 cells, T24 cells and UMUC-3 cells which were treated with ETS-1 over-expression vector. These data

ETS-1 do not control cell proliferation in bladder cancer
To explore whether ETS-1is associated with cell proliferation in bladder cancer, the bladder cancer cell lines treated with ETS-1 shRNA or over-expression vector were analyzed by the CCK-8 assay and EdU assay. Both of the two results indicated that there was no difference in the proliferation rate between ETS-1 shRNA or over-expression vector group and negative control group in 5637 cells ( Figure 5A, 5D, 5G and 5J), T24 cells ( Figure 5B, 5E, 5H and 5K) and UMUC-3 cells ( Figure 5C, 5F, 5I and 5L). These results confirmed that ETS-1is not associated with the bladder cancer cell proliferation.

ETS-1 do not regulate cell apoptosis in bladder cancer
Finally, we determined whether ETS-1 inhibit cell apoptosis in bladder cancer. At 48 hours after transfection of ETS-1 shRNA or transduction of the over-expression vector, the relative activity of caspase-3 of the bladder cancer 5637 cells, T24 cells and UMUC-3 cells were detected by thecaspase-3 enzyme-linked immunosorbent assay (ELISA) assay. Regrettably, as shown in Figure  6A-6C, there was also no difference in the relative activity of caspase-3 between ETS-1 shRNA and negative control shRNA transfected group in these three bladder cancer cells. As shown in Figure 6C-6E, there was also no difference in the relative activity of caspase-3 between ETS-1over-expression vector and negative control transduced group in these three bladder cancer cells. The results indicated that ETS-1 has nothing to do with the bladder cancer cell apoptosis.

DISCUSSION
ETS-1, one of the ETS transcription factor members, is known to play important roles in a number of physiological and pathological processes, such as embryonic development, angiogenesis, and hematopoietic differentiation [3,4]. For example, ETS-1 is essential for Connective Tissue Growth Factor (CTGF/CCN2) induction mediated by TGF-β1 in Osteoblasts [8] and ETS-1 is significant for EBNA3Cmediated up-regulation of AK-B transcription, which leads to degradation of p53 [9]. Furthermore, ETS-1 is expressed in many tumors and activates many cancer related genes that promote the malignant transformation and progression of tumors, including invasion, metastasis and neoangiogenesis [10][11][12]. In gastric tumors [13] and oral squamous cell carcinomas [14], the deregulation of ETS-1 was related with the invasive behavior and cancer metastasis. In addition, ETS-1was responsible for the malignant progression and invasion of meningiomas and human astrocytic tumor [15,16]. Over-expression of ETS-1was also reported in human breast cancer and angiosarcoma of the skin [17,18].
In our study, we found that the expression level of ETS-1 was significantly up-regulated in bladder cancer tissues compared with matched normal tissues. These results in our research manifested that ETS-1 is likely to play oncogenic role in bladder cancer. To further understand the biological functions of ETS-1 in bladder cancer, the ETS-1 specific shRNA and over-expression vector were designed Oncotarget 25131 www.impactjournals.com/oncotarget to treat bladder cancer cells, and then we used scratch assay and invasion assay to detect the cell migration ability and the cell invasiveness. As expected, ETS-1 promoted cell migration and cell invasion in the related bladder cancer cell lines. In addition, cell proliferation and cell apoptosis were tested by the CCK-8, EdU and ELISA assay to verify whether ETS-1 influences the cell proliferation and cell apoptosis in bladder cancer. Unfortunately, there were no differences in both the cell proliferation rate and the relative activity of caspase-3 between the treated groups in bladder cancer cells. These findings indicate that ETS-1 may only play vital roles in the migration and invasion of bladder cancer, and did not affect the proliferation and apoptosis.
It should be noted that the results of the correlation of ETS-1 expression with the malignant phenotype of bladder cancer in our study is contrary to the findings from Aysegul S, et al. They reported that most of the low-grade and noninvasive bladder cancer largely expressed ETS-1, and that high-grade and invasive bladder cancer showed a frequent decrease or loss of ETS-1expression [19]. But they only used immunohistochemical staining to investigate the expression of ETS-1 and did not perform the functional experiments in their studies. The expression of ETS-1 was not associated with grade of bladder cancer (P = 0.202) ( Table 2) in our study. More clinic samples should be needed to reveal the correlation of ETS-1 expression pattern with the clinical-pathological characteristics in bladder cancer.
In conclusion, the results of this research demonstrate that ETS-1 is highly expressed in the bladder cancer compared to matched normal bladder tissue, and promotes bladder cancer cell migration and invasion. Hence, ETS-1 should play oncogenic roles in human bladder cancer and it can be used as a therapeutic target for treating human bladder cancer. The future works are still needed to study the molecular mechanisms of ETS-1 which maybe a candidate biomarker for bladder cancer in the clinic.

Patient samples
42 patients with bladder urothelial carcinomas were included in the study. Bladder cancer tissue and paired normal bladder tissue from each patient were snap-frozen in liquid nitrogen instantly after partial or radical cystectomy. Written informed consents were obtained from all the patients. The clinical-pathological characteristics of the patients are summarized in Table 2. The study was approved by the Institutional Review Board of Shenzhen Second People's Hospital.

Cell lines and culture conditions
Bladder cancer T24, 5637 and UMUC-3 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). T24 cell line and UMUC-3 cell line were cultured in Dulbecco's Modified Eagle Medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C in a 5% CO 2 atmosphere. 5637 cell line was cultured in RPMI-1640 Medium (Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C in a 5% CO 2 atmosphere.
Bladder cancer cells were cultured in 6-well plates (2 × 10 5 /well) and infected with the lentivirus at a multiplicity of infection (MOI) of 20 for 24 h. Cells were selected using puromycin (Sigma-Aldrich, St. Louis, MO, USA) for getting stable ETS-1expression. At last, ETS-1 expression in the infected cells was confirmed by means of qRT-PCR.

Total RNA extraction and reverse transcription
Total RNA was extracted from the tissue samples, the transfected cells and the infected cells by using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. cDNA was converted from total RNA by using the PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa, Otsu, Shiga, Japan) according to the instructions. www.impactjournals.com/oncotarget

Cell migration assay
The treated cells were seeded on the 6-well plates (5 × 10 5 /well). At about 90% confluent cells, a clean line was created by using a sterile 200 μl pipette tip at 5 h post transfection. The migration of cells was monitored using a digital camera system and imaged at the time of 0 h and 16 h. The cell migration distance (mm) was calculated using the software program HMIAS-2000. Experiments were performed at least three times.

Cell invasion assay
About 1 × 10 5 treated cells with 100 μl serum-free medium were plated into the upper chambers (24-well insert, pore size 8 μm, Corning) which were added with Matrigel (1:8, 50 μl/well, BD Bioscience, San Jose, CA, USA) . Concurrently, the lower chambers were filled with 500 μl medium containing 10% fetal bovine serum. Cells were cultured at 37°C in a 5% CO 2 atmosphere for 48 hours. Cells under the surface of the lower chamber were washed with 1 × PBS, fixed with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet for 25 min, and then washed 3 times. Invaded cells were observed under the inverted microscope and imaged. Afterwards, each chamber with the invaded cells was soaked into 1 ml 33% acetic acid for 10 min to wash out the crystal violet. 100 μl/ well 33% acetic acid were added into 96-well plates, and the absorbance was measured at a wavelength of 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Experiments were performed in triplicate.

Cell proliferation assay
The effects of down-regulation or up-regulation ETS-1 on cell proliferation were examined by Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China) according to the previous studies [27,28]. The treated cells were seeded in a 96-well plate (5 × 10 3 /well) and cultured in normal medium. At 0, 24, 48, and 72 h after transfection, 15 µl of CCK-8 was added into each well of 96-well plate and the cells were cultured for 1 hour. Absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Assays were repeated at least three times.
Cell proliferation was also tested by 5-ethynyl-2-deoxyuridine (EdU) incorporation assay using the corresponding kit (Ribobio, Guangzhou, China) .The EdU incorporation assay was carried out according to the previous studies [29,30]. Finally, the cells were observed using fluorescence microscopy. All experiments were performed in three times.

Cell apoptosis assay
The effects of down-regulation or up-regulation ETS-1 on cell apoptosis were detected by calculating the activity of caspase-3 using the Caspase-3 enzyme-linked immunosorbent assay (ELISA) assay kit (Hcusabio, Wuhan, China) according to the manufacturer's instructions. Absorbance was measured by using a microplate reader (Bio-Rad, Hercules, CA, USA). Data were shown as the ratios between the absorbance of experimental groups and those of negative control groups. Each test was carried out at least three times.

Statistical analyses
All experimental data from three independent experiments were presented as mean ± standard deviation (SD). All statistical data were analyzed by SPSS 19.0 software (SPSS Inc.Chicago, IL, USA). The ETS-1 RNA expression differences between bladder cancer tissue and matched normal tissue were analyzed using paired samples t-test. The relationship between ETS-1 level and clinicalpathologic characteristics in 42 patients with bladder cancer were analyzed by Continuous correction chi-square test. The data of CCK-8 assay were analyzed by ANOVA and independent samples t-test was used to analyze other data. A P value of less than 0.05 was considered to be statistically significant.