The present study was approved by the Ethics Committee of Central South University (Changsha, China). Informed consent was obtained from each of the patients in the study. Eighteen bladder cancer tissues, in addition to their matched adjacent normal tissues, were collected in the Department of Urinary Surgery, Xiangya Hospital of Central South University. Tissue samples were immediately frozen in liquid nitrogen following surgical removal.

Human bladder cancer T24 cells were obtained from the Cell Bank of Central South University, and cultured in Dulbecco's modified Eagle's medium (Gibco Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (Gibco Life Technologies) at 37°C in a humidified incubator containing 5% CO₂.

Total RNA was extracted using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. For the analysis of miRNA expression, TaqMan® MicroRNA Reverse Transcription kit (Invitrogen Life Technologies) was used to convert RNA into cDNA, in accordance with the manufacturer's instructions. qPCR was then performed using All-in-One™ miRNA qPCR Detection kit (GeneCopoeia, Rockville, MD, USA) on an ABI 7500 thermocycler (Applied Biosystems, Foster City, CA, USA). The PCR cycling conditions were set as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 15 sec. U6 was used as a reference gene. The relative expression was analyzed by the 2^−ΔΔCt method.

Tissue or cell solubilization was performed using cold radioimmunoprecipitation lysis buffer. The proteins were separated with 12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane, which was subsequently incubated with Tris buffered saline with Tween 20 containing 5% milk at room temperature for 3 h. The PVDF membrane was then further incubated with rabbit polyclonal anti-Cdc42 (1:100; cat. no. ab64533), rabbit monoclonal anti-phospho-STAT3 (1:50; cat. no. ab76315), rabbit monoclonal anti-STAT3 (1:50; cat. no. ab32500) and rabbit monoclonal anti-GAPDH (1:50; cat. no. ab181602) primary antibodies (Abcam, Cambridge, UK), respectively, at room temperature for 3 h. Subsequent to being washed three times in phosphate-buffered saline (PBS) with Tween 20, the membrane was incubated with the goat anti-rabbit secondary antibody (Abcam) at room temperature for 40 min. Chemiluminescent detection was carried out using an enhanced chemiluminescence kit (Pierce Chemical, Rockford, IL, USA), and the relative protein expression was analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Relative protein expression was shown as the density ratio versus GAPDH.

Transfection was performed using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer's instructions. For the functional analysis of miR-195, bladder cancer T24 cells were transfected with scrambled miRNA as a negative control, miR-195 mimics or miR-195 inhibitor (Invitrogen Life Technologies). For the functional analysis of Cdc42, human bladder cancer T24 cells were transfected with Cdc42-specific small interfering RNA (siRNA) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or pcDNA3.1-Cdc42 plasmid (Nlunbio, Changsha, China).

A mutant-type 3′-UTR of Cdc42 was produced using the QuikChange™ Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). The wild- and mutant-type 3′-UTRs of Cdc42 were inserted into the psiCHECK™2 vector (Promega Corp., Madison, WI, USA), respectively. Human bladder cancer T24 cells were cultured to ~60% confluence in a 24-well plate, and the T24 cells were then transfected with psiCHECK™2-Cdc42-3′-UTR or psiCHECK™2-mutant Cdc42-3′-UTR vector, respectively, with or without 100 nm miR-195 mimics, using Cellfectin® II Reagent (Invitrogen Life Technologies). After 48 h the dual-luciferase activities in each group were examined on an LD400 luminometer (Beckman Coulter, Fullerton, CA, USA). The activity of Renilla luciferase was normalized to that of firefly luciferase.

T24 cells in each group were seeded into a 96-well plate. Following culture for 48 h, MTT (0.5 µg/ml) was added into the medium for 1 h of incubation. The medium was then removed and the plate was washed three times in PBS, prior to the addition of 100 µl dimethylsulfoxide to dissolve the precipitate. The absorbance at 570 nm was determined using a microplate reader (Bio-Rad, Hercules, CA, USA).

The results are expressed as the mean ± standard deviation of three independent experiments. The statistical analysis of differences was performed using one-way analysis of variance with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The expression level of miR-195 was determined in bladder cancer tissues, as well as their matched normal adjacent tissues, using RT-qPCR. As shown in Fig. 1A, miR-195 expression in the bladder cancer tissues was notably downregulated compared with that in the matched adjacent tissues. The expression of miR-195 was further examined in bladder cancer T24 cells, which showed that miR-195 expression was also downregulated in T24 cells compared with that in normal tissues.

The protein level of Cdc42 was determined in bladder cancer tissues and their matched adjacent normal tissues. Western blotting data showed that the protein level of Cdc42 was upregulated in bladder cancer tissues and T24 cells compared with that in normal tissues (Fig. 1B), suggesting that Cdc42 may play a suppressive role in bladder cancer.

Bioinformatics prediction was performed using TargetScan (http://www.targetscan.org/), which showed that the putative seed sequences for miR-195 at the 3′-UTR of Cdc42 were highly conserved (Fig. 2A). The wild and mutant types of Cdc42 3′-UTR were then generated, and dual-luciferase reporter assays were performed in bladder cancer T24 cells in order to verify whether Cdc42 is a direct target of miR-195. As shown in Fig. 2B, the Renilla/firefly value of luciferase activity was notably decreased only in bladder cancer T24 cells co-transfected with the wild-type 3′-UTR of Cdc42 and miR-195 mimics, indicating that Cdc42 is a direct target of miR-195 in bladder cancer T24 cells.

To further determine the effects of miR-195 on Cdc42 expression in bladder cancer cells, bladder cancer T24 cells were transfected with miR-195 mimics, miR-195 inhibitor and scramble miRNA, respectively. As shown in Fig. 3, the protein level of Cdc42 was notably decreased in T24 cells transfected with miR-195 mimic, while increased in T24 cells transfected with miR-195 inhibitor, indicating that miR-195 negatively regulates the protein level of Cdc42 in bladder cancer T24 cells.

As Cdc42/STAT3 signaling has been found to largely contribute to bladder cancer cell proliferation (14), and miR-195 can inhibit the protein expression of Cdc42 in bladder cancer T24 cells, we speculated that miR-195 could play a suppressive role in bladder cancer cell proliferation, via downregulation of Cdc42/STAT3 signaling. To verify this theory, bladder cancer T24 cells were transfected with miR-195 mimics or Cdc42-specific siRNA, or co-transfected with miR-195 mimics and Cdc42 plasmid, respectively. A cell proliferation assay was then performed. It was found that upregulation of miR-195 and the inhibition of Cdc42 significantly inhibited T24 cell proliferation. In addition, the inhibitory effect of miR-195 upregulation on T24 cell proliferation was significantly reversed by the overexpression of Cdc42 (Fig. 4A). It was further confirmed that the activation of STAT3 was inhibited by miR-195 upregulation (Fig. 4B). Accordingly, we suggest that miR-195 inhibits bladder cancer cell proliferation at least partially by the inhibition of Cdc42/STAT3 signaling.