BC tissues (n=67) and pair-matched noncancerous tissues were obtained through tissue biopsy from patients diagnosed with BC at the Department of Urology, Yidu Central Hospital of Weifang between March 2013 and September 2016. Informed consent was obtained from patients. All procedures involving human participants were in accordance with the ethical standards of the Human Research Ethics Committee at the Department of Urology, Yidu Central Hospital of Weifang.

Bladder cancer SW780, UMUC3, 5637, T-24 and one normal urothelial cell line SVHUC-1 utilized in present study were purchased from the Tumor Cell Bank of the Chinese Academy of Medical Science (Shanghai, China). The UMUC3, T24 and SV-HUC-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) plus 10% fetal bovine serum and ampicillin and streptomycin at 37°C in a humidified atmospherewith 95% air and 5% CO₂. The 5637 and SW780 cells were cultured in RPMI-1640 medium (Invitrogen Life Technologies) plus 10% fetal bovine serum and ampicillin and streptomycin at 37°C in a humidified atmosphere with 95% air and 5% CO₂.

Total RNAs from tissues and cells were isolated with TRIzol reagent (Invitrogen Life Technologies) under the manufacturer's instructions. Reverse transcription was performed with PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's instructions. RT-qPCR was performed with SYBR Prime Script RT-PCR kits (Takara Bio, Inc.) based on the manufacturer's instructions. The SNHG5 level was calculated with the 2^−ΔΔCt method, which was normalized to GAPDH mRNA. The primers for SNHG5 were as the following: forward, 5′-CGCTTGGTTAAAACCTGACACT-3′ and reverse, 5′-CCAAGACAATCTGGCCTCTATC-3′; the primers for GAPDH were as the listed: Forward, 5′-ACGGGAAGCTCACTGGCATGG-3′ and reverse, 5′-GGTCCACCACCCTGTTGCTGTA-3′. All assays were performed in triplicate. The expression levels were relative to the fold change of the corresponding controls, which were defined as 1.0.

Cells were transfected with siRNAs for SNHG5 by using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer's instructions. After transfection (48 h), cells were harvested for the following experiments including RT-qPCR, western blot analysis, proliferation assays and flow cytometry. RNA oligonucleotides were purchased from GenePharma (Shanghai, China). The siRNA sequence for SNHG5 was si-SNHG5, 5′-CCTCTGGTCTCATCTGCATATTGACTTA-3′.

Cell viability was assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-trtrazolium bromide (MTT) assay. 5×10³ cells/well transfected with indicated vector were seeded in a 96-well flat-bottomed plate for 24 h and cultured in a normal medium. At 0, 24, 48, 72 and 96 h after transfection, the MTT solution (5 mg/ml, 20 µl) was added to each well. Following incubation for 4 h, the media was removed and 100 µl of DMSO was added to each well. The relative number of surviving cells was assessed by measuring the optical density (OD) of cell lysates at 560 nm. All assays were performed in triplicate.

Cells (500 cells/well) transfected with indicated vector were plated in 6-well plates and incubated in RPMI-1640 with 10% FBS at 37°C. Two weeks later, the cells were fixed and stained with 0.1% crystal violet. The number of visible colonies was counted manually.

Apoptosis was performed by using flow cytometric analysis with Annexin V: FITC Apoptosis Detection kits (BD Biosciences, San Diego, CA, USA), according to the manufacturer's instructions. For cell cycle distribution, cells were collected directly or 48 h after transfection and washed with ice-cold phosphate-buffered saline (PBS), and fixed with 70% ethanol overnight at −20°C. Fixed cells were rehydrated in PBS for 10 min and incubated in RNase A (1 mg/ml) for 30 min at 37°C, then the cells were subjected to PI/RNase staining followed by flow cytometric analysis with a FACScan instrument and CellQuest software (both from Becton-Dickinson, Mountain View, CA, USA) as described (20).

Total protein lysates were separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and were electrophoretically transferred to polyvinylidene difluoride membranes (Roche Diagnostics, Indianapolis, IN, USA). Protein loading was estimated by using mouse anti-GAPDH monoclonal antibody. The membranes were blotted with 10% non-fat milk in TBST for 2 h at room temperature, washed and then probed with the rabbit anti-human p27 (1:2,000 dilution), CDK2 (1:2,000 dilution), activated caspase-3 (1:2,000 dilution), activated caspase-9 (1:2,000 dilution), and GAPDH (1:3,000 dilution), overnight at 4°C, followed by treatment with secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature. The proteins were detected by using an enhanced chemiluminescence system and then exposed to x-ray film. All antibodies were purchased from Abcam (Cambridge, MA, USA).

Data were shown as the means ± standard error of at least three independent experiments. The SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Two group comparisons were performed with a Student's t-test. Multiple group comparisons were analyzed with one-way ANOVA. The Pearson χ² test was used to evaluate the relationship between SNHG5 expression and clinical features. Kaplan-Meier method was used to compare the overall survival curves between high-SNHG5 and low-SNHG5 expression groups via the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

To explore the biological function of SNHG5 in BC, we first measured the level of SNHG5 in BC tissues and corresponding normal tissues (n=67) by RT-qPCR. As shown in Fig. 1A, SNHG5 was aberrantly increased (P<0.01) in tumor tissues compared with that in corresponding normal tissues. Furthermore, the levels of SNHG5 in four bladder cancer cells SW780, UMUC3, 5637, T-24 and one normal urothelial cell line SVHUC-1 were assessed. As presented in Fig. 1B, the level of SNHG5 was significantly increased in four BC cell lines in comparison to that in the normal urothelial cell line. And among these cell lines, the expression level of SNHG5 in UMUC3 and 5637 cell was relative higher than that in SW780 and T-24 cells; therefore, we chose UMUC3 and 5637 as the study object in the following assays. These data revealed that SNHG5 may play a pivotal role in BC progression.

Then we investigated the relationship between SNHG5 expression and clinicopathological features in BC, the mean expression level of SNHG5 in all BC tissues was used as a cutoff value, and all samples were divided into two groups (high expression group, n=36 vs. low expression group, n =31). As illustrated in Table I, high expression level of SNHG5 was significantly correlated with larger tumor range (P=0.001), metastasis (P=0.013), lymph nodes (P=0.001) and pathological stage (P=0.003), but it had no significant correlation with age, sex and smoking (P>0.05). Furthermore, Kaplan-Meier method analysis (log-rank test) was performed to determine the association between SNHG5 expression and overall survival of patients. As shown in Fig. 2, patients with high expression level of SNHG5 had a significantly shorter overall survival than those with low level of SNHG5 (P=0.000).

To investigate the biological function of SNHG5 on the proliferation of BC cells, UMUC3 cell and 5637 cell were transfected with si-SNHG5 andsiRNA was used as as negative control (NC). The satisfactory transfection efficiency was obtained at 48 h post-transfection (Fig. 3A). MTT assay was performed to measure the function of SNHG5 on cell viability. As shown in Fig. 3B, weakened proliferation ability was obtained from UMUC3 cell and 5637 cell transfected with si-SNHG5 in comparison to the NC-transfected cells. Consistent with the results of MTT, colony formation assay revealed a growth-inhibition effect mediated by si-SNHG5 (Fig. 3C). The findings indicated that silenced SNHG5 could suppress the proliferation of BC cells.

To investigate the underlying mechanism of si-SNHG5-mediated growth-inhibition, flow cytometric analysis of cell cycle distribution were performed. As illustrated in Fig. 4A, silenced SNHG5 in UMUC3 cell and 5637 cell obviously induced cell cycle arrest at G1. And flow cytometric analysis of apoptosis revealed that silenced SNHG5 significantly increased the apoptosis rate of UMUC3 cell and 5637 cell (Fig. 4B). Furthermore, western blot assay demonstrated that the level of cyclin-dependent kinase 2 (CDK2) was decreased while the level of p27 was significantly increased; and apoptosis-related proteins (activated caspase-3 and activated caspase-9) were increased when SNHG5 was knockdown (Fig. 4C). These data indicated that SNHG5 contributed to the proliferation ability of BC cells, which might be attributed to its influence on cell cycle and apoptosis.

To determine whether p27 was involved in the si-SNHG5-mediated growth inhibition, we first measured the level of p27 in BC tissues and corresponding normal tissues by RT-qPCR and then western blot analysis was performed to determine the protein level of p27 in four pairs of cancer tissues and normal tissues. As illustrated in Fig. 5A and B, the mRNA level of p27 was significantly downregulated in BC tissues and the protein level was also obviously decreased in BC tissues. Furthermore, rescue assays were performed to verify whether SNHG5 regulated BC cell proliferation via silencing p27 expression. UMUC3 and 5637 cells were co-transfected with si-SNHG5 and si-p27, and results from MTT and colony-formation assays revealed that co-transfection with si-p27 could partially abolish the si-SNHG5-mediated growth-inhibition (Fig. 5C). Additionally, flow cytometric analyses of apoptosis and cell cycle distribution showed that co-transfection with si-p27 could partially rescue the si-SNHG5-mediated cell cycle arrest and increased apoptosis rate (Fig. 5D and E). These results indicated that the effect of SNHG5 on BC was partially involved with targeting p27.