The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases provide an invaluable resource of publicly available gene expression data that can be integrated and analyzed to derive new hypothesis and knowledge. In this study, the difference in Rab6c expression between tumor and normal of bladder cancer samples were identified in the Gene Expression Omnibus (GEO) database (ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3167) (25) and the database included 41 tumor tissue samples from bladder cancer patients and 9 normal tissue samples. The data was unpaired and had no gender distribution information. In addition, miR-218 expression in bladder cancer samples was analyzed from Cancer Genome Atlas (TCGA) database (portal.gdc.cancer.gov/) (26) and the database included 412 tumor tissue samples from bladder cancer patients and 19 normal tissue samples. The data was paired but there was no gender distribution information. In addition, TargetScan human 7.1 (targetscan.org/vert_72/) was used to analyze the target genes of miR-218 in bladder cancer.

Tumor tissue and matched adjacent normal tissue samples were collected from 6 patients with bladder cancer (aged 55–67 years) undergoing surgery at the General Hospital of Shenyang Military from 2008/1/31 to 2014/3/31 (Shenyang, China). The inclusion criteria were male patients diagnosed with bladder cancer by histology or cytology. The exclusion criteria were those who had received systemic anti-cancer treatment for metastatic or persistent/recurrent disease, or had a disease involving the bladder during screening. Notably, <3 cm is adjacent normal tissue, 3–5 cm is near cancer tissue, and greater than 5 cm is distant cancer tissue (27). Permission for the collection and application of patient samples was granted by the Ethics Committee of the General Hospital of Shenyang Military. In addition, each patient signed a written informed consent before operation. All obtained clinical tissue specimens were immediately frozen in liquid nitrogen and stored at −80°C.

Bladder cancer cell lines (SV-HUC-1, T24 and EJ) were purchased from the American Type Culture Collection and cultured for 24 h at 37°C in a humidified atmosphere containing 5% CO₂ using DMEM (HyClone; Cytiva), containing 10% FBS (HyClone; Cytiva) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). All T24 and EJ cells were authenticated by short tandem repeat and confirmed negative for mycoplasma contamination prior to the experiments. The cells were transferred to the second generation for lentivirus transfection. Lentivirus vectors plasmid were constructed by GenePharma. Following the instructions of Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), recombinant miR-218 lentivirus particles (1×10⁸ TU/ml) constructed with 5 µg GV309, Rab6c lentivirus particles (1×10⁸ TU/ml) constructed with 7.5 µg plent-EF1a-FH-CMV-GP were used to transfected cultured cells (5×10⁵) using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) in DMEM with 10% FBS at a multiplicity of infection of 10 for 20 min. The lentiviral vector with green fluorescent protein (GFP) and resistance tag (Puromycin, 400 ng/ml) was used to select the cells. 72 h after transfection, the expression of GFP of the cells were observed under fluorescence microscope (×200 magnification; Olympus Corporation) and cultured for 24 h at 37°C in a humidified atmosphere containing 5% CO₂ for subsequent experiments.

TRIzol reagent (Invitrogen, Germany) was used to obtain RNA from bladder cancer tissues and cells. According to the manufacturer's instructions, a TaqMan MicroRNA reverse transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for reverse transcription of total RNA to obtain cDNA to analyze miRNA expression. Subsequently, template cDNA was used for qPCR analysis with PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and miR-218 or GAPDH primers, in which GAPDH was used as an internal reference. mRNA expression analysis of Rab6c was performed in the same manner. The thermocycling conditions were as follows: Initial denaturation 95°C, 15 min; 40 of cycles of 55°C for 15 min and final extension at 85°C for 2 min. The primer sequences used in this study were as follows: Rab6c sense, 5′-AGGAGATCTGCCGCCGCGATCGC-3′ and antisense, 5′-CGAGCGGCCGCGTACGCGTCCTC-3′; miR-218 sense, 5′-CGAGTGCATTTGTGCTTGATCTA-3′ and antisense, 5′-TGGTGTCGTGGAGTCG-3′; U6 sense, 5′-CTCGCTTCGGCAGCACA-3′ and antisense, 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH sense, 5′-ACAACTTTGGTATCGTGGAAGG-3′ and antisense, 5′-GCCATCACGCCACAGTTTC-3′. The relative mRNA expression of miR-218 and Rab6c was quantified with cycle threshold values and normalized using the 2^−∆∆Cq method (28). U6 was the internal control of miR-218 expression, and GAPDH was the internal control of Rab6c expression. The expression of miR-218 and Rab6c was relative to the fold change of the corresponding negative controls, which was defined as 1.0.

Total protein was extracted from cultured cells or tissues using RIPA lysis buffer (Beyotime). Subsequently, protein content was determined by the bicinchoninic acid method (BCA, Pierce; Thermo Fisher Scientific, Inc.) and 30 µg of protein from each group was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Bioscience). After the membranes were blocked using TBST solution containing 5% skimmed milk and 0.5 ml/l Tween-20 at 4°C for 1 h, incubated with primary antibody against Rab6c (1:500; Invitrogen; Thermo Fisher Scientific, Inc.; PA5-39409) and GAPDH (1:1,000; Santa Cruz Biotechnology, sc-47724) at room temperature for 2 h, they were incubated with horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology) with 1:5,000 dilution. The protein signal bands were visualized using an enhanced chemiluminescence detection reagent (ECL; Thermo Scientific, Inc.) and analyzed by ImageJ software 1.8.0.112 (National Institutes of Health).

The T24 and EJ bladder cancer cell lines were seeded at 2×10³ cells/well and cultured in 6-well plates for 5 days. The manufacturer's instructions of the CCK-8 kit (Dojindo) were strictly followed to perform the cell counting experiment using a microplate reader (BioTek) to measure the optical density (OD) at 450 nm.

The colony formation assay was performed as previously described (29). Briefly, transfected T24 and EJ bladder cancer cells were cultured in 6-well plates at a density of 1,000 cells/well for 10 days. After the cell colonies were treated with methanol for 15 min, they were stained with 0.1% crystal violet for 10 min at room temperature. After the number of cells in a single clone was greater than 50, and the size between 0.3–1.0 mm, we started counting and taking pictures under an optical microscope (Olympus). The percentage of colony formation was calculated by setting the control group to 100%.

Briefly, 2×10⁴ transfected T24 and EJ cells were seeded in the upper Transwell invasion chambers (24-well, 8-mm pore; Corning), which were coated with Matrigel (BD Biosciences) at 37°C. The lower chamber was filled with medium containing 10% FBS. After 48 h, the unmigrated cells were removed, and the cells that migrated to the bottom were fixed with 70% ethanol and stained with 0.1% crystal violet for 20 min at room temperature. Next, the stained cells were photographed under fluorescent microscope (200× magnification, Olympus Corporation) and counted by ImageJ software 1.8.0.112 (National Institutes of Health).

The sequences of h-Rab6c-3′UTR-wild-type (WT) or h-Rab6c-3′UTR-mutant (Mut) were synthesized, connected into the pSI-Check2 vector (Hanbio) and extracted plasmids. The 293T cells (Chinese Academy of Sciences, Shanghai, China) were cultured in 96-well plates for 24 h at 37°C until the cell density reached 5×10⁴ cells/ml and co-transfected with the corresponding plasmids (0.16 µg) with Lipofectamine 2000 (0.3 µl, 0.8 mg/ml; Invitrogen;; Thermo Fisher Scientific, Inc.) at 37°C. After 6 h of transfection, the cells were exchanged for fresh DMEM medium, and cultured for 48 h at 37°C for subsequent Renilla luciferase detection. Follow the instructions of the Dual Luciferase Reporter Assay Kit (Promega Corporation), 100 µl Passive Lysis Buffer was added to the 96-well plate and centrifuged at 1,200 × g at 4°C for 10 min, and then 100 µl Luciferase Assay Reagent II and 100 µl cell lysate were added in sequence and mixed by pipetting 2–3 times. 100 µl STOP & GLO^® reagent (Luciferase Assay Reagent; Promega Corporation) was added and mixed 2–3 times to record the Renilla luciferase value, which was the reporter gene luminescence value.

The presented results were representative of experiments repeated at least three times and all data as the mean ± standard deviation (SD). Statistical analysis was conducted with SPSS 21.0 (IBM, Inc.) and GraphPad 8.0 (GraphPad Software). All tests were analyzed using paired t-test and one-way ANOVA followed by Bonferroni's post hoc test analysis. P<0.05 was considered to indicate a statistically significant difference.

Previous studies have reported that miR-218 was associated with the development of a variety of cancers, including bladder cancer (23,30). In addition, Rab6c is a member of the Rab family and is involved in drug resistance in MCF7 cells (31). The relationship between Rab6c and miR-218 in bladder cancer cell lines was examined in the current study. First, the difference in Rab6c expression between tumor and normal of bladder cancer samples was identified in the GEO database. The results indicated that the expression level of Rab6c in tumor tissues was significantly higher compared with that in normal tissues (Fig. 1A). Conversely, TCGA database results demonstrated that miR-218 expression was significantly lower in bladder cancer tissues compared with that in normal tissues (Fig. 1B).

Subsequently, the expression levels of Rab6c and miR-218 were detected in clinical samples of bladder cancer. As expected, Rab6c mRNA expression was higher in tumor tissues compared with in normal tissues (Fig. 1C). At the same time, it was identified that miR-218 was expressed at low levels in bladder cancer (Fig. 1D). Consistently, Rab6c protein expression was abnormally elevated in tumor tissue compared with normal tissues (Fig. 1E). Due to the individual differences and pathological grade stages of these 6 patients with bladder cancer, the expression level of Rab6c varied among different patients. In fact, in patients with advanced bladder cancer (cases 2, 3, 6), Rab6c was expressed in normal tissues adjacent to the cancer, but it is still lower than that in tumor tissues. In addition, Rab6c expression in T24 and EJ cells was relatively higher compared with that in SV-HUC-1 cells (Fig. 1F). In general, Rab6c was upregulated in bladder cancer, while miR-218 was expressed at low levels.

Rab6c and miR-218 overexpressing cells were constructed to examine their effects in bladder cancer cells. As presented in Fig. 2A, miR-218 overexpression was established in T24 and EJ cells. It was found that Rab6c protein expression was decreased in miR-218-overexpressing T24 and EJ cells (Fig. 2B). Moreover, the expression level of miR-218 was downregulated in Rab6c-overexpressing T24 and EJ cells (Fig. 2C).

According to the conserved miR-218 binding site (-AAGCACAA-) in the 3′UTR of Rab6c mRNA, as indicated by TargetScan, a publicly available algorithm, Rab6c was preliminarily identified as a promising target for miR-218 (Fig. 3A). Compared with the NC group, hsa-miR-218-3p significantly downregulated the luciferase activity in h-Rab6c-3′UTR-wt group, indicating an interaction between RAB6C and miR-218. However, hsa-miR-218-3p failed to downregulate luciferase activity in h-Rab6c-3′UTR-mu group (Fig. 3B and C). These results suggested that there was a negative regulatory effect between Rab6c and miR-218 in bladder cancer.

As the biological function of Rab6c in bladder cancer cells has not been revealed, to the best of our knowledge, proliferation was evaluated in T24 and EJ cells using CCK-8 and colony formation assays. The result of western blotting indicated that Rab6c expression was significantly upregulated following transfection with Rab6c lentivirus particles (Fig. 4A). The CCK-8 assay demonstrated that cell proliferation was notably promoted by Rab6c overexpression (Fig. 4B). Consistent with the results, Rab6c overexpression accelerated colony formation, as indicated by an increased number of colonies (Fig. 4C). To elucidate whether the effects of Rab6c overexpression were reversed by miR-218, restoration experiments were performed. As shown in Fig. 4B and C, the cells overexpressing Rab6c and miR-218 exhibited a lower proliferation rate and fewer colonies compared with the cells overexpressing Rab6c alone.

Bladder cancer cell invasion was examined using a Transwell assays. Rab6c overexpression significantly promoted the invasion of bladder cancer cells (Fig. 5). However, co-transfection with miR-218 overexpression significantly reduced the invasion of T24 and EJ cells. Thus, it was concluded that Rab6c may serve a stimulative role in the proliferation and invasion of bladder cancer cells, which could be reversed by miR-218.