Human BCa T24, UM-UC-3, SW780, HT1376, RT4 and J82 cell lines, immortalized human bladder epithelium SV-HUC-1 cells and the 293 cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured according to the manufacturer's instructions at 37°C in an atmosphere of 5% CO₂. Total mRNA and miRNA were independently extracted from the cells using RNAzol RT RNA Isolation Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) and stored at −20°C for subsequent analysis.

The expression of miR-124 was quantified using an All-in-one miRNA qPCR kit (GeneCopoeia, Inc., Rockville, USA) and U6 was used as the control. The primers used for the RT-PCR were: miR-124 (forward 5′-GCGGCCGTGTTCACAGCGGACC-3′ and reverse 5′-GTGCAGGGTCCGAGGT-3′) and U6 (forward 5′-CGCTTCGGCAGCACATATACTA-3′ and reverse 5′-CGCTTCACGAATTTGCGTGTCA-3′). To determine the mRNA expression of STAT3, the total RNA in T24 cells was reverse transcribed into cDNA at 37°C using Eastep RT Master Mix kit (Promega Corporation, Madison, WI, USA), then STAT3 mRNA was quantified using SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Japan) and GAPDH was used as the control. The thermocycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 34 sec, then 72°C for 45 sec. The primers used for qPCR were: STAT3 (forward 5′-ATCACGCCTTCTACAGACTGC-3′ and reverse 5′-CATCCTGGAGATTCTCTACCACT-3′) and GAPDH (forward 5′-CCACTCCTCCACCTTTGAC-3′ and reverse 5′-ACCCTGTTGCTGTAGCCA-3′). Relative expression was calculated using the 2^−ΔΔCq method (16).

T24 cells were centrifuged in 1,000 × g for 5 min at 37°C subsequent to the addition of 1 ml ice-cold PBS and washed three times with ice-cold PBS and lysed using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology, Haimen, China) for 30 min at 4°C. The cell lysate protein was centrifuged at 15,000 × g at 4°C for 10 min and the protein was quantified using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). A total of 20 µg protein/lane was separated by 10% SDS-PAGE, and the resolved proteins were transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked using 5% skim milk powder at 4°C overnight and then hybridized with the primary antibodies targeting the proteins including GADPH (1:2,500; cat no. ab9485), STAT3 (1:1,000; cat no. ab68153), vascular endothelial growth factor receptor (VEGFR; 1:1,000; cat no. ab11939), B-cell lymphoma 2 (Bcl-2; 1:1,000; cat no. ab32124), B-cell lymphoma-extra large (Bcl-xl; 1:1,000; cat no. ab32370), MCL1, Bcl2 family apoptosis regulator (Mcl-1; 1:2,000; cat no. ab32087), Cyclin D1 (1:10,000; cat no. ab134175) and MYC proto-oncogene, BHLH transcription factor (cMyc; 1:1,000; cat no. ab32072) (all from Abcam, Cambridge, UK) at 4°C overnight. Subsequent to washing using PBS with 0.05% Tween-20 three times at 5 min each time, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibody Goat Anti-Rabbit immunoglobulin G H&L (1:2,000; cat no. ab6721; Abcam) conjugated to horseradish peroxidase at 4°C overnight. Results were quantified by the Molecular Imager ChemiDoc XRS+ System 2.0 (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH was used as the control.

The whole 3′UTR of the STAT3 gene was cloned and amplified in 293 cells. TargetScan 6.2 bioinformatics prediction software (http://www.targetscan.org/; date of access, 20 February 2017) was used to predict their binding sites in 20 February, 2017. The conserved sites of miR-124 were selected and then target gene STAT3 was selected for sites in its 3′UTR. A mutation in the 3′UTR of the STAT3 gene at the putative binding site of miR-124 was generated with the QuickChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc. Santa Clara, CA, USA). The wild and mutant STAT3 genes were cloned into the pmirGlO-vector (Promega Corporation) immediately downstream of the Renilla luciferase gene. Cells were co-transfected with pmirGlO-3′UTR-STAT3 (50 ng), miR-124 (50 nM), or scramble mimic (50 nM) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell lysates were prepared using Passive Lysis Buffer (Promega Corporation) 48 h after transfection. The luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega Corporation) and normalized to Renilla luciferase activity.

T24 (1×10⁵ cells/plate) were plated in 6-well plates at 37°C overnight and transfected with miR-124 mimics or NC mimics (20 nM; GeneCopoeia, Inc.) using Lipofectamine^® 2000. The miR-124 mimic sequence used was: Forward, 5′-GCTCTAGAGGCCTCTCTCTCCGTGTTCCACAGCGGACCTTGATTTAAATGTCCATACAATTAAGGCACGCGGTTGAATGCCAAGAATGGGGCTG-3′ and reverse, 5′-CGGGATCCCAGCCCCATTCTTGGCATTCACCGCGTGCCTTAATTGTATGGACATTTAAATCAAGGTCCGCTGTGAACACGGAGAGAGAGGCCT-3′. Following transfection for 6 h at 25°C, the Opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.) without serum was changed with the fresh medium. Then cells were assayed by RT-qPCR and western blot analysis for each group according to the aforementioned protocols following culture for 48 h.

Small interfering RNA Target Finder online design software (Ambion; Thermo Fisher Scientific, Inc.) was used to select a segment (5′-AAGAGTCAAGGAGACATGCAA-3′, 670–690 bp) in the coding region of STAT3 (GenBank serial no. NM139276) as a target sequence. Then, the corresponding DNA template strands containing restriction sites for HindIII and BamH I were designed for the target sequences. The template strand sequences were as follows: Sense, 5′-GATCCGAGTCAAGGAGACCATGCAATTCAAGAGATTGCATGTCTCCTTGACTCTTTTTTGG-3′; and anti-sense, 5′-AGCTTTTCCAAAAAAGAGTCAAGGAGACATGCAATCTCTTGAATTGCATGTCTCCTTGACTCG-3′. The shRNA sequences were obtained by PCR amplification, and vector plasmid pGENESIL (Wuhan Genesil Biotechnology Co., Ltd., Wuhan, China) were digested and connected by HindIII and BamH I enzymes (Thermo Fisher Scientific, Inc.). The recombinant plasmid (pGENESIL-shRNA-STAT3) was amplified and infected (0.8 µg plasmid, at 25°C for 20 min) into the T24 cells. After 6 weeks of G418 (800 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc.) selection, stably-transfected cells (STAT3-shRNA) were used for subsequent experiments.

The adherent cells were digested at 37°C with 0.25% trypsin (Bioswamp; Wuhan Myhalic Biotechnological Co., Ltd., Wuhan, China) and centrifuged at 1,000 × g at 25°C for 5 min. The supernatant was discarded and the cells were washed three times with PBS. The cells (1–5×10⁵) were collected and resuspended in 200 µl binding buffer (cat no. C1052-1; Beyotime Institute of Biotechnology) for analysis using the Cell Cycle and Apoptosis Analysis kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Analyses were performed using a LSRII Flow Cytometry System equipped with FACSDiva software 4.1 (BD Biosciences, Franklin Lakes, NJ, USA). Data was analyzed with the ModFit LT software package version 4.0 (Verity Software House, Inc., Topsham, ME, USA).

The WST-1 Cell Proliferation and Cytotoxicity Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol was used to analyze cell viability and cell proliferation at 0, 24, 48, 72 and 96 h. All experiments were performed in triplicate. For the colony formation assay, cells were plated at a density of 200 cells/well in standard culture conditions (5% CO₂ and 37°C) for 14 days. The colonies were fixed with 5 ml 4% absolute methanol for 15 min at 25°C, stained at 37°C with 0.1% crystal violet for 20 min and washed three times. Then a light microscope (TS-100F; Nikon, Tokyo, Japan) was used to visualize the results at a magnification of ×40.

T24 cells were seeded (1×10⁶ cells/plate) in a 6-well plate. Wounding of the confluent cell growth was performed by scratching with a 10 µl pipette tip 24 h after cell plating. The cells were then cultured for 24 h and wound closure was detected by inverted microscopy (magnification, ×200; Olympus, Tokyo, Japan). The experiment was repeated three times.

Statistical analysis was conducted using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Unpaired Student's t-tests were used to analyze the results. Pearson's linear correlation analysis was used to analyze the association between the expression of STAT3 and miR-124. All experiments were performed in triplicate and the data are expressed as mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the function of STAT3 in human BCa, the expression of STAT3 was detected in 6 human BCa cell lines (T24, UM-UC-3, SW780, HT1376, RT4 and J82) and immortalized human bladder epithelium SV-HUC-1 cells. STAT3 was overexpressed as an oncogene in the BCa cell lines compared with SV-HUC-1 cells. The expression of STAT3 was highest in T24 cells (Fig. 1A). The association between miR-124 and STAT3 in the BCa cell lines was additionally evaluated by RT-qPCR. Notably, there was a significant inverse correlation between miR-124 and STAT3 (Fig. 1B). From these results, it was hypothesized that miR-124 is a potent miRNA regulator of STAT3 expression. T24 cells, which overexpressed STAT3 and exhibited low expression of miR-124, were selected for subsequent experiments.

The effects of the transfection of miR-124 mimics on STAT3 mRNA and protein expression levels were determined by RT-qPCR and western blot analysis. There was an ~80% decrease in STAT3 mRNA expression and a 60% decrease in STAT3 protein expression in T24 cells transfected with miR-124 mimics (Fig. 1C and D). This suggested that STAT3 may be downregulated by miR-124. Post-transcriptional regulation by miRNAs involves binding to the 3′UTR of the relevant downstream genes (17). To additionally define the association of miR-124 with STAT3, Target Scan 6.2 bioinformatics prediction software (http://www.targetscan.org/) was used to predict their binding sites. The results revealed that the miR-124 seed-matching sequence in STAT3 3′UTR was ‘CACGGAA’ (Fig. 1E). Luciferase reporter assays demonstrated that the presence of miR-124 resulted in a marked decrease in luciferase activity when the STAT3 plasmid containing wild type 3′UTR was present, while luciferase activity did not change significantly when the mutant 3′UTR was present (Fig. 1F). These data suggested that miR-124 may target STAT3 transcription, potentially contributing to the downregulation of STAT3 expression.

To additionally investigate the role of miR-124/STAT3 signaling pathway in BCa, a stable cell line with low STAT3 expression (STAT3-shRNA-T24) and a control cell line (shRNA-control-T24) were constructed (Fig. 2A). The result demonstrated that STAT3 mRNA and protein levels in the STAT3-shRNA-T24 were significantly inhibited (Fig. 2B and C). Flow cytometry was conducted to confirm that the knockdown of STAT3 suppressed the growth of T24 BCa cells. A significant increase in numbers of cells in the G1 phase was observed in STAT3-shRNA-T24 cells compared with that in shRNA-control-T24 cells. This increase in G0/G1 cell population was accompanied with a concomitant decrease in cell numbers in S and G2-M phases in the STAT3-shRNA-T24 cells (Fig. 3A). A WST-1 assay was then performed to investigate whether STAT3 exhibited a biological function in the proliferation of T24 cells. A significant inhibition in STAT3-shRNA-T24 was evident (Fig. 3B). Consistent with the result from the WST-1 assay, the colony formation assay revealed markedly fewer and smaller STAT3-shRNA-T24 colonies compared with the shRNA-control-T24 cell line (Fig. 3C).

To investigate the effect of STAT3 on the migration of T24 BCa cells, the capacity of STAT3-shRNA-T24 cells to migrate was measured using a wound healing assay. STAT3-shRNA-T24 cells exhibited slower rates of wound closure and decreased numbers of stained cells in the wound healing assay compared with the shRNA-control-T24 cells (Fig. 4A). Furthermore, a marked induction of apoptosis was confirmed in STAT3-shRNA-T24 cells (Fig. 4B). The results demonstrated that the silencing of STAT3 significantly suppressed the migratory capability and induced apoptosis of T24 cells. Finally, the downstream target genes of STAT3 following the knockdown of STAT3 were examined using western blot analysis (Fig. 4C). Notably, cyclin D1, cMyc, Bcl-2, Bcl-xl, Mcl-1 and VEGFR were decreased in the STAT3-shRNA-T24 cells. Taken together, these results demonstrate that STAT3 knockdown repressed cell proliferation, migration, invasion and vasculogenic mimicry, indicating the oncogenic role of STAT3 in BCa.