Introduction

Bladder cancer is one of the most common malignancies in the urinary tract, with an average of 350,000 new cases and 145,000 deaths every year worldwide [1]. Moreover, there has been a rising incidence of bladder cancer recently [2]. Despite development of various surgical and chemotherapeutic methods for treating bladder cancer, the 5-year survival rate is still relatively low due to the high recurrence and metastasis of bladder cancer [3]. Therefore, identification of key genes involved in the pathogenesis of bladder cancer is of importance for the development of novel therapeutic strategies against this malignancy.

MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs of ∼22 nucleotides. They regulate a large number of target genes, typically via enhancement of mRNA degradation or inhibition of its translation [4–7]. MiRNAs are implicated in the regulation of many biological processes, including cell proliferation, differentiation, migration, invasion, and apoptosis. Several lines of evidence indicate that miRNAs are frequently dysregulated in human cancers including bladder cancer [8–35]. MiRNAs affects many aspects of tumorigenesis by acting as an oncogene or tumor suppressor. Previous studies have suggested miR-192 as a tumor suppressor in several types of human cancers such as colon cancer [36] and renal cell cancer [37]. In contrast, in some other types of cancers such as pancreatic ductal adenocarcinoma [38], miR-192 plays a favorable role in tumor growth and progression. A recent study has reported that miR-192 is downregulated in patients with bladder cancer relative to healthy controls [39]. However, the mechanism view of this downregulation of miR-192 in bladder cancer is lacking.

Therefore, in the present study, we employed gain-of-function approaches to investigate the roles of miR-192 in the proliferation, cell cycle progression, and apoptosis of bladder cancer cells.

Materials and methods

Cell culture

Human bladder cancer lines HT-1376, 5637, TCCSUP, ScaBER, UM-UC-3, SW780, J82, T24, RT4, and HT-1197, as well as normal uroepithelial SV-HUC-1 cells were all purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). They were cultured in RPMI 1640 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified atmosphere of 5 % CO2 at 37 °C. Data from T24 cells were shown.

Transfection of miR-192

Human bladder cancer cells were seeded at a density of 3 × 105 cells/well onto 6-well plates and allowed to attach overnight. Cells were then transfected with 50 nM of either human miR-192 precursor or non-specific control miRNA (Ambion Austin, TX, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Transfection efficiency was estimated to be >85 %, which was determined by co-transfection of fluorescence-labeled control small interfering RNA (siRNA; Shanghai GenePharma, Shanghai, China). After 24-h incubation, cells were given fresh media with 10 % FBS and incubated for additional 48–72 h before cell viability and apoptosis measurement.

Measurement of miR-192

Total RNA was isolated from cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The level of mature miR-192 was measured using Taqman miRNA Assays (Applied Biosystems, Foster City, CA, USA). Briefly, cDNA synthesis was done using a miRNA-specific stem-loop primer, and quantitative PCR was performed using specific TaqMan MicroRNA Assay primers. Real-time quantitative RT-PCR (qRT-PCR) analysis was carried out using Applied Biosystems 7500 Real-Time PCR System. The relative miR-192 amount normalized to the internal control U6 small nuclear RNA was calculated with the comparative cycle threshold (ΔΔCt) method.

Cell proliferation assay

Human bladder cancer cells were seeded onto 96-well plates in triplicate at a density of 1 × 103 cells/well 24 h after transfection with miR-192 precursor or non-specific control miRNA. Cells were cultured for further 48 or 72 h and cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Briefly, MTT solution (5 mg/mL; Sigma, St. Louis, MO, USA) was added to the cell cultures and incubated at 37 °C for 4 h. After removal of the MTT solution, dimethyl sulfoxide (DMSO) solution was added to dissolve formazan crystals. The absorbance at 570 nm was measured.

Cell cycle analysis

Human bladder cancer cells were transiently transfected with miR-192 or control miRNA, and at 48 h after transfection, cells were harvested and stained for 15 min with 0.05 mg/mL propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA) in the presence of 0.02 mg/mL RNase H. Cell cycle distribution was analyzed by flow cytometry.

Cell apoptosis assay

At 48 h after transfection with miR-192 or non-specific control miRNA, human bladder cancer cells were harvested, washed, and resuspended. Cell apoptosis was measured using the Annexin V Apoptosis Kit following the manufacturer’s instructions (Becton Dickinson Biosciences, San Diego, CA, USA). The cells were stained with fluorescein isothiocyanate-conjugated Annexin V and PI solution (20 μg/mL) for 15 min in the dark. Apoptotic cells (annexin V-positive) were analyzed by flow cytometry.

Western blot analysis

At 48 h after transfection with miR-192 or control miRNA, human bladder cancer cells were lysed in ice-cold radioimmunoprecipitation assay buffer containing 1 mM phenylmethanesulfonyl fluoride and complete protease inhibitors (Roche, Mannheim, Germany). Equal amount of proteins (∼50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking the non-specific binding sites, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies used in this study included mouse anti-cyclin D1, anti-p21, anti-p27, anti-Bcl-2, anti-Bax, anti-Mcl-1, and anti-α-tubulin monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) for 1 h. Protein bands were visualized with a chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) and analyzed using the Quantity One software (Bio-Rad, Hercules, CA, USA).

Statistical analysis

Data were expressed as means ± standard deviation (SD). Statistical significance was evaluated by Student’s t test or one-way analysis of variance (ANOVA) followed by the Tukey’s test. Differences were considered statistically significant at p < 0.05.

Results

Enforced expression of miR-192 suppresses T24 cell proliferation

RT-qPCR analysis revealed that the miR-192 expression level was significantly lower in T24 human bladder cancer cells than normal uroepithelial cells (Fig. 1a). This result confirmed the downregulation of miR-192 in bladder cancer. To determine the biological role of miR-192 in bladder cancer cells, here, we overexpressed miR-192 precursor in T24 cells and investigated the effect of restoration of miR-192 on bladder cancer cell proliferation. We found that enforced expression of miR-192 decreased the proliferation of T24 cells by 22 and 54 % at 48 and 72 h after culturing, respectively (p < 0.05 compared to control miRNA-transfected cells, Fig. 1b).

Fig. 1
figure 1

Enforced expression of miR-192 suppresses bladder cancer cell proliferation. a The expression of miR-192 in T24 bladder cancer cells and normal uroepithelial SV-HUC-1 cells was measured by qRT-PCR analysis. *P < 0.05. b MTT assay was done to determine the viability of T24 cells transfected with miR-192 precursor or control miRNA at 48 and 72 h. *P < 0.05 vs. control miRNA

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Effect of overexpression of miR-192 on cell cycle distribution

Next, we checked whether the growth-suppressive effect of miR-192 overexpression may be associated with induction of cell cycle arrest. Flow cytometry analysis showed that T24 cells with miR-192 overexpression exhibited a significant (p < 0.05) increase in G0/G1 phase and a significant decrease in S phase, compared to control miRNA-transfected cells (Fig. 2). These results suggest a G0/G1 cell cycle arrest induced by miR-192 overexpression.

Fig. 2
figure 2

Effect of overexpression of miR-192 on cell cycle distribution of T24 cells. Cells were transfected with miR-192 precursor or control miRNA and stained with PI before flow cytometry analysis. *P < 0.05 vs. control miRNA

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Effect of miR-192 on cell cycle control genes

Next, we examined the effect of miR-192 overexpression on several important genes involved in cell cycle control. Western blot analysis demonstrated that miR-192 overexpression resulted in a 3–6-fold increase in the expression levels of p21 and p27 and decrease in the expression level of cyclin D1 (p < 0.05 compared to control cells; Fig. 3).

Fig. 3
figure 3

Western blot analysis of p21, p27, and cyclin D1 in T24 cells transfected with miR-192 precursor or control miRNA. Densitometric data were expressed as fold change relative to control miRNA-transfected cells assigned a value of 1. *P < 0.05 vs. control miRNA

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Overexpression of miR-192 promotes apoptotic death of T24 cells

We also checked the effect of overexpression of miR-192 on T24 cell apoptosis. Annexin-V/PI staining assay demonstrated that overexpression of miR-192 significantly induced apoptotic death in T24 cells compared to the delivery of control miRNA (18.2 ± 2.3 % vs. 3.5 ± 1.2 %, P < 0.05; Fig. 4).

Fig. 4
figure 4

miR-192 promotes apoptotic death of T24 cells. Cells were transfected with miR-192 precursor or control miRNA and 48 h later, apoptosis was assessed using annexin-V/PI staining assay. *P < 0.05 vs. control miRNA

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Effect of miR-192 on Bcl-2 family proteins

Since the Bcl-2 protein family plays a key role in the regulation of apoptosis in a wide range of cells, we studied the effect of miR-192 overexpression on the expression of Bcl-2 family proteins. Western blot analysis showed that miR-192 overexpressing T24 cells had a significant (p < 0.05) increase in Bax (a pro-apoptotic protein) and a significant decrease in Bcl-2 and Mcl-1 (two anti-apoptotic proteins) compared to control cells (Fig. 5).

Fig. 5
figure 5

Western blot analysis of Bax, Bcl-2, and Mcl-1 in T24 cells transfected with miR-192 precursor or control miRNA. Densitometric data are expressed as fold change relative to control miRNA-transfected cells assigned a value of 1. *P < 0.05 vs. control miRNA

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Discussion

Aberrant regulation of miRNAs has been described in many previous studies, which is considered as an important mechanism for carcinogenesis [8–35]. Several miRNAs such as miR-99a [10, 40, 41], miR-29c [42], and miR-490-5p [43] are downregulated in bladder cancer, acting as a negative regulator of tumor growth and development. Previous reports also show that miR-192 expression is significantly reduced in patients with bladder cancer. Here, we confirmed the downregulation of miR-192 in bladder cancer cells compared to non-malignant bladder epithelial cells. These findings suggest that miR-192 may function as a tumor suppressor in bladder cancer. Indeed, our functional studies revealed that miR-192 overexpression impaired the proliferation and induced apoptosis in several bladder cancer cells. Although we only showed data on T24 cells, we got very similar results from other lines that had been examined, which suggests a cell-line independent result of our study.

Our findings are consistent with several previous studies that have shown that restoration of miR-192 exerts inhibitory effects on tumor cell proliferation and invasion. However, in some other types of cancers, miR-192 shows tumor-promoting activities. For instance, Zhao et al. reported that overexpression of miR-192 promotes tumor growth and progression in human pancreatic ductal adenocarcinoma [38]. MiR-192 also contributes to gastric cancer progression as evidenced by the finding that inhibition of miR-192 significantly suppressed gastric cancer cell invasion [44]. Taken together, miR-192 plays distinct roles in different types of cancers and acts as a tumor suppressor in bladder cancer.

Induction of cell cycle arrest is an important mechanism for the action of tumor suppressor genes. Jiang et al. reported that the growth-suppressive effect of miR-16 in bladder cancer cells is associated with negative regulation of cyclin D1 [44]. Lin et al. demonstrated that targeting cyclin-dependent kinase 4 mediates the induction of G1-phase cell cycle arrest by miR-195 in bladder cancer cells [24]. MiR-192 overexpression led to profound G2/M arrest in proximal tubular epithelial cells. In agreement with studies, our data revealed that restoration of miR-192 in bladder cancer cells resulted in a cell cycle arrest at the G0/G1 phase. Moreover, we found that miR-192 overexpression significantly decreased the expression of cyclin D1 and increased the expression of p21 and p27. Cyclin D1 is a nuclear protein required for cell cycle progression in G1 [45]. Cyclin D1 downregulation has been reported to mediate the anticancer effect of isorhapontigenin on human bladder cancer cells [46]. The cyclin-dependent kinase (CDK) inhibitors p21 and p27 can bind to and inhibit the activity of various cyclin-CDK complexes, thus blocking S phase entry [47]. These studies suggest that miR-192-induced G0/G1 cell cycle arrest in bladder cancer cells is causally linked to the deregulation of cyclin D1, p21, and p27.

In addition to the induction of cell cycle arrest, miR-192 overexpression caused apoptotic death in bladder cancer cells. Western blot analysis revealed that the protein level of Bax was increased, and the protein levels of Bcl-2 and Mcl-1 were decreased in miR-192-overexpressing T24 cells. The Bcl-2 protein family, which comprises both pro- and anti-apoptotic members, plays a critical role in the regulation of apoptotic cascades [48]. Bax can translocate from the cytosol to mitochondria in response to pro-apoptotic stimuli, resulting in activation of mitochondrial apoptosis. The anti-apoptotic protein Bcl-2 has the ability to block Bax-induced apoptosis by binding to Bax. Mcl-1 promotes cell survival by interfering at an early stage in the apoptotic pathway leading to release of cytochrome c from mitochondria [49]. Our data indicate that miR-192 overexpression-induced apoptosis of human bladder cancer cells is largely associated with alteration of the Bcl-2 family proteins.

To summarize, restoration of miR-192 suppresses the proliferation and induces cell cycle arrest at the G1 phase and apoptosis in bladder cancer cells. The growth inhibitory effect of miR-192 overexpression is likely mediated through the regulation of a large number of genes involved in cell cycle control and apoptosis. Therefore, miR-192 appears to be a promising therapeutic target in bladder cancer treatment.