Prior written informed consent was obtained from all patients and the study was approved by the Protection of Human Subjects Committee of XiangYa Hospital of Central South University (Changsha, China).

A total of 9 BC tissue samples and adjacent non-tumorous kidney tissue counterparts were used for RT-qPCR and western blot analysis and were collected at XiangYa Hospital of Central South University. The hard and firm tumor tissues were trimmed free of normal tissue and were immediately snap frozen in liquid nitrogen. All BC cases were clinically and pathologically confirmed to be bladder carcinoma.

The human bladder epithelial cell line, SV-HUC-1, and the bladder tumor cell lines T24, 5637, 3-UM-UC-3 and RT4 were obtained from the Cell Bank of Central South University (Changsha, China). Cells were cultured in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) with 10% FBS (Invitrogen Life Technologies), 50 U/ml of penicillin and 50 mg/ml of streptomycin (Invitrogen Life Technologies). All cells were cultured in a sterile incubator maintained at 37°C with 5% CO₂.

Total RNA was extracted from cells with TRIzol reagent (Invitrogen Life Technologies) following the manufacturer's instructions. The relative expression level of miR-128 was determined by RT-qPCR using mirVana™ qRT-PCR microRNA detection kit (Ambion Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. Specific primer sets for miR-128 (HmiRQP3056) and U6 (HmiRQP9001) were obtained from Genecopoeia, Inc. (Rockville, MD, USA). The expression levels of VEGF-C mRNA was detected by RT-qPCR using the standard SYBR Green RT-PCR Kit (Takara Bio, Inc., Otsu, Japan) following the manufacturer's instructions. The specific primer pairs are as follows: VEGF-C, sense 5′-CACGAGCTACCTCAGCAAGA-3 and antisense 5′-GCTGCCTGACACTGTGGTA-3′; and β-actin as an internal control, sense 5′-AGGGGCCGGACTCGTCATACT-3′ and antisense 5′-GGCGGCACCACCATGTACCCT-3′. The relative expression levels of VEGF-C mRNA or miR-128 were quantified using GraphPad Prism software, version 4.0 (GraphPad Software, Inc., San Diego, CA, USA) and the 2^−ΔΔCt method (29).

Western blot analysis was performed as described previously (13). Protein levels were quantified by Bradford assay (30). A total of 30 mg protein from each sample was fractionated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (PVDF membranes; EMD Millipore, Billerica, MA, USA). The membrane was blocked in 0.1% Triton X-100 (Invitrogen Life Technologies) and 5% low fat milk powder (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline for 1 h at 4°C and then probed with rabbit polyclonal primary anti-β-actin (1:500, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or rabbit polyclonal primary anti-VEGF-C (1:500, Immunoway, USA). After washing 3 times with Tris-buffered saline Tween-20 (Sigma-Aldrich, the membrane was incubated in peroxidase-conjugated goat anti-mouse/rabbit IgG antibody (1:1,000, ImmunoWay Biotechnology, Co., Newark, DE, USA). The bands were visualized by an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA) using medical X-ray films (Kodak, Rochester, NY, USA) and quantified by Photoshop software (Adobe Systems, San Jose, CA, USA). The intensities of the bands of interest were expressed relative to the β-actin intensities from the same sample.

For the miR-128 functional analysis, the pre-miR-128, pre-control [negative control (NC) of pre-miRNA], anti-miR-128 or anti-control (NC inhibitor) (Genecopoeia, Inc.) constructs were transfected into T24 and 5637 cell lines using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer's instructions.

Cells in exponential growth were seeded at a final concentration of 2×10³ cells/well in 96-well plates. The viability of the cells was evaluated by MTT assay (Invitrogen Life Technologies) after 24, 48 and 72 h of seeding. The optical density at 570 nm (OD₅₇₀) of each well was measured with an ELISA reader (ELX-800 type, BioTek Instruments, Inc., Winooski, VT, USA).

For all groups, 3 ml complete medium containing 150 cells were added to each well of a 6-well plate. The plates were incubated at 37°C, 5% CO₂ for 14 days. The cells were then gently washed and stained with Giemsa (Invitrogen Life Technologies). Colonies containing ≥50 cells (0.3–1.0 mm) were counted.

The invasive ability of bladder cancer cells was then studied in 24-well transwell chambers coated with matrigel (EMD Millipore). For all groups, 200 µl of 1×10⁶ cells/ml cell suspension was added in triplicate wells. After a 24-h incubation, the dye on the membrane was dissolved with 10% acetic acid, dispensed into 96-well plates (150 µl/well), and the OD₅₇₀ of each well was measured with an ELISA reader (ELX-800 type; BioTek Instruments, Inc.).

The cell migratory capability was estimated using a wound healing assay as described previously (31). In brief, cells were cultured to confluence. Wounds of approximately 1 mm width were created with a plastic scriber and cells were washed and incubated in a serum-free medium. After wounding, the cells were incubated for 24 h in a medium containing 10% fetal bovine serum. Cultures at 0 and 48 h were observed under an inverted microscope (TS100; Nikon Corporation, Tokyo, Japan).

The 3′-UTR of VEGF-C (NM_005429.4) containing the miR-128 binding sites and its corresponding mutated sequence were cloned into the psi-CHECK2 luciferase reporter vector (Promega Corporation, Madison, WI, USA) downstream of Renilla luciferase, named VEGF-C-3′-UTR and VEGF-C-Mut 3′-UTR, respectively. Using Lipofectamine 2000 (Invitrogen Life Technologies), T24 and 5637 cells were co-transfected with the reporter constructs and miR-128 mimics, the miR-128 inhibitor, NC or the NC inhibitor. Luciferase activity was determined after 48 h using the Dual-Glo substrate system (Promega Corporation) and a Beckman Coulter LD400 luminometer (Beckman Coulter, Inc., Brea, CA, USA). Data are presented as the ratio of experimental (Renilla) luciferase to control (Firefly) luciferase.

Data are expressed as the mean ± standard deviation from >3 separate experiments. Statistical analysis was performed using SPSS software, version 15.0 (SPSS, Inc., Chicago, IL, USA). The difference between 2 groups was analyzed by the Student's t-test. A value of P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression levels of miR-128 and VEGF-C in clinical tissues or in BC cell lines were determined using RT-qPCR. Compared with normal adjacent tissues, the mRNA expression levels of miR-128 in tumor tissues were significantly reduced compared with in normal tissues (Fig. 1A; P=0.0017), while the mRNA expression levels of VEGF-C were reduced (Fig. 2A; P=0.013). Meanwhile, the downregulation of miR-128 and upregulation of VEGF-C were also observed in BC cell lines compared with SV-HUC-1 (Figs. 1B and 2B; P=0.004 and P=0.003, respectively). The VEGF-C protein expression was apparently increased in tumor tissues or BC cell lines compared with adjacent normal tissues or SV-HUC-1 as assessed by western blot analysis (Fig. 2C and D; P=0.0023 and P=0.015, respectively). These results indicate that miR-128 may serve an important role in malignant progression of BC. Furthermore, the coexistence of VEGF-C upregulation and miR-128 downregulation in BC cells implies a potential regulatory association between the 2 molecules.

To clarify the functions of miR-128 in BC cells, gain-of-function and loss-of-function cell models were constructed by transfection with pre-miR-128 and anti-miR-128. The miR-128 mRNA level was significantly upregulated (Fig. 3A and B; P=0.034 and P=0.037, respectively), while VEGF-C level was downregulated when transfected with pre-miR-128 (Fig. 3C and D; P=0.042 and P=0.039, respectively). By contrast, when transfected with anti-miR-128, the mRNA level of miR-128 was significantly downregulated (Fig. 3A and B; P=0.034 and P=0.037, respectively) and VEGF-C was significantly upregulated (Fig. 3C and D; P=0.042 and P=0.039, respectively). The results indicated that overexpression of miR-128 reduced the VEGF-C expression in T24 and 5637 cells (Fig. 3E; P=0.044).

To assess whether VEGF-C is a direct target of miR-128, luciferase reporter assays were applied. The VEGF-C 3′-UTR fragment containing the miR-128 binding site and mutated targeting sequence were cloned into psi-CHECK2 dual luciferase reporter vectors. The miR-128 signifcantly inhibited the luciferase activity in both T24 and 5637 cells transfected with the VEGF-C-3′-UTR. However, miR-128 mimics did not suppress the luciferase activity levels in the T24 and 5637 cells transfected with Mut-VEGF-C-3′-UTR (Fig. 4A and B; P=0.024 and P=0.027, respectively). These findings indicate that VEGF-C is a direct target of miR-128.

To investigate the effects of miR-128 on proliferation of BC cells, T24 and 5637 cells were transfected with miR-128 mimics or its inhibitor. MTT and colony formation assays were used to demonstrate that overexpression of miR-128 markedly reduced the growth (Fig. 5A and B; P=0.0017 and P=0.015, respectively) and clone-formation (Fig. 5C and D; P=0.035 and P=0.038, respectively) rate of the 2 BC cell lines as compared with that of negative control (NC) transfected cells. However, inhibition of miR-128 increased the growth rate of the 2 BC cell lines as compared with that of NC transfected cells. These results indicated that miR-128 inhibited the proliferation of BC T24 and 5637 cells.

To explore the functional role of miR-128 on migration and invasion in BC cells, T24 and 5637 cells were transfected with pre-miR-28 and anti-miR-128, respectively. Wound healing and transwell assays were performed to evaluate the cell metastasis capacity. The results demonstrated that cell migration and invasion capacity were significantly increased when the BC cells were transfected with pre-miR-128, while cell migration and invasion capacity were significantly reduced when cells were transfected with anti-miR-128 (Fig. 6C, P=0.037 and P=0.026, T24 and 5637 cells, respectively; Fig. 6D, P=0.031; Fig 6E, P=0.035). The results indicated that miR-128 supressed the migration and invasion of bladder cancer T24 and 5637 cells.