Human BC cells of the 5637, J82 and T24 cell lines, and the SV-HUC-1 human immortalized uroepithelium cell line, were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology (Shanghai, China). 5637, J82 and T24 cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and SV-HUC-1 cells were cultured in F-12K medium (Gibco; Thermo Fisher Scientific, Inc.). Media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified incubator with 5% CO₂. Cells of the 5637 and T24 cell line were exposed to 60 or 80 ng/ml celecoxib (PeproTech, Inc., Rocky Hill, NJ, USA) for 48 h at 37°C in an atmosphere containing 5% CO₂. Cells treated with dimethyl sulfoxide alone were used as controls.

Cells of the 5637 and T24 cell line were plated in 6-well plates at a density of 2×10⁵ per well. The has-miR-145-5p inhibitor, mimic and negative control (NC) were obtained from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequence of miR-145-5p mimics and miR-145-5p inhibitors was 5′-GUCCAGUUUUCCCAGGAAUCCCUGGAUUCCUGGGAAAACUGGACUU-3′ and 5′-AGGGAUUCCUGGGAAAACUGGAC-3′, respectively, and that of the negative control was 5′-UUC UCC GAA CGU GUC ACG UTT-3′. Target Scan Human 7.2 (www.targetscan.org/vert_72) was used to identify the predicted target of miR-145-5p (17). When the 5637 and T24 cells were grown to 60-70% confluence, the cells were transfected with 20 or 40 nM miR-145-5p mimics or 40 nM inhibitors using Lipofectamine™ 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and Opti-MEM medium (Gibco; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 6 h following transfection, the medium was replaced with fresh medium containing 10% FBS. Cells were then incubated for 48 h at 37°C in an atmosphere containing 5% CO₂.

At 48 h following transfection or celecoxib treatment, total RNA was extracted from T24 and 5637 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min at room temperature. cDNA was synthesized using a Takara PrimeScript RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol and specific miR-145 primers from the Bulge-Loop™ hsa-miR-145-5p RT-qPCR Primer Set (Guangzhou RiboBio Co., Ltd.) were used for miRNA quantitative analysis. RT-qPCR was performed using a SYBR Premix Ex Taq kit (Takara Bio, Inc., Otsu, Japan) in the ABI PRISM 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using 400 ng total RNA. PCR reaction conditions were performed as follows: 95°C for 2 min, followed by 40 cycles at 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. The RT-qPCR results were normalized against an internal control (U6 for miR-145, GAPDH for mRNA) and the relative expression levels were evaluated using the 2^−∆∆Cq method (18) and then expressed as the fold change. All reactions were performed at least in triplicate. The oligonucleotide sequences of the RT-qPCR primers are listed in Table I.

Total proteins from the 5637 and T24 cells were prepared using RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China). Proteins were quantified using a Pierce BCA protein Assay kit (Thermo Fisher Scientific, Inc.), followed by western blot analysis. Proteins from cell lysates (40 µg) were subjected to 12% SDS-PAGE. Once the blots were transferred to a polyvinylidene difluoride membrane, they were blocked for 2 h in 5% skimmed milk in Tris-buffered saline with Tween^®-20 at room temperature. Subsequently, antibodies targeting GAPDH (cat no. ab8245; 1:1,000; Abcam, Cambridge, UK), COX-2 (cat no. 12282; 1:1,000; CST Biological Reagents Co., Ltd., Shanghai, China) Vimentin (cat no. ab32131; 1:500; Abcam), E-cadherin (cat no. 3195; 1:1,000; CST Biological Reagents Co., Ltd.), Smad3 (cat no. 9523; 1:1,000; CST Biological Reagents Co., Ltd.) and TGFBR2 (cat no. 79424; 1:1,000; CST Biological Reagents Co., Ltd.) were used to incubate the membranes overnight at 4°C, followed by a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat no. ab6721; 1:5,000; Abcam; 1.5 h incubation at room temperature). Protein bands were visualized by enhanced chemiluminescence (cat. no. WBKLS0500; EMD Millipore, Billerica, MA, USA). Image J 1.45 software (National Institutes of Health, Bethesda, MD, USA) was used to perform densitometry analysis of each band.

The effects of celecoxib on the viability of 5637 and T24 cells were assessed using a Cell Counting Kit-8 (CCK-8) detection kit (Nanjing Keygen Biotech Co., Ltd., Nanjing, China). Cells in the exponential growth phase were seeded into a 96-well plate at a density of 6,000 cells per well. After 24 h, celecoxib (0-300 µM) was added to the medium. The cells were incubated at 37°C for 48 h, then CCK-8 solution was added and the plate was incubated at 37°C for 2.5 h. Absorbance was measured at a wavelength of 450 nm with a TECAN SPARK 10M Microplate Reader (Tecan Group, Ltd., Männedorf, Switzerland). Cell viability was expressed as a percentage of absorbance in the treated wells compared with that of the untreated (control) wells. Each experiment was performed in triplicate and repeated at least three times.

Cell migration was determined using a wound healing assay. Cells of the 5637 and T24 cell lines were treated with celecoxib, transfected with miR-145 mimic or non-target control for 24 h, and then seeded in 6-well plates. A wound (~300 µm) was made by scratching the monolayer with a 10 µl pipette tip. The wounded monolayer was then washed three times with phosphate buffered saline to remove cell debris and treated with celecoxib for 24 h. Subsequent to scratching, the area of the cell-free scratch was photographed using a Olympus CKX41 inverse light microscope (Olympus Corporation, Tokyo, Japan) at 0 and 24 h. Cell invasion capacities were measured using a Transwell assay (Corning Incorporated, Corning, NY, USA). The Transwell assay was performed using a 24-well Transwell chamber (pore size, 8 µm; Corning Incorporated). Cells of the 5637 and T24 cell lines were treated with celecoxib and/or transfected with miR-145 inhibitor, mimic or negative control for 24 h. Subsequently, a total of 8×10⁴ cells/well were seeded in the upper chamber of the Transwell system. The upper chamber of the insert was precoated with 0.1 ml (300 µg/ml) Matrigel matrix (Corning Incorporated) for the invasion assay. The invaded cells on the underside of the membrane were then counted. In this assay, prepared cells were seeded in the upper chamber with serum-free RPMI-1640 medium and the medium of the lower chamber was supplemented with 10% FBS as a chemoattractant. Following incubation for 24 h, the cells were fixed using 4% formaldehyde at 37°C for 30 min. Cells not invading through the pores were removed using a cotton swab. Cells that had invaded to the lower surface of the membrane were stained using crystal violet at room temperature for 15 min. Finally, five representative fields at a magnification of ×100 were randomly imaged and quantified for each well using the Olympus CKX41 inverse light microscope.

All data are presented as the mean ± standard deviation in at least three replicates per group. Statistical analysis was performed to determine the significance of the difference between groups using one-way analysis of variance followed by post hoc Turkey's honest significant difference test, or a Student's t test. All statistical analyses were performed using GraphPad Prism 7.00 software (GraphPad Software, Inc., La Jolla, CA, USA) for Windows. P<0.05 was considered to indicate a statistically significant difference.

To investigate the function of celecoxib in BC cells, two BC cell lines, 5637 and T24, were selected to undertake cell viability, wound healing and Transwell assays. As presented in Fig. 1A, consistent with the results of a previous study, celecoxib inhibits the growth of 5637 and T24 cells (10). Additionally, following stimulation with 60 µM celecoxib for 24 h, there were significantly fewer cells invading to the lower surface compared with the control group (P<0.05; Fig. 1B) and less wound healing compared with the control group (Fig. 1C). The present study then evaluated the expression of COX-2 in 5637 and T24 cells following celecoxib treatment. As presented in Fig. 2A, celecoxib downregulated the expression of COX-2. Furthermore, it was detected that the mRNA and protein expression levels of the EMT-associated molecule, E-cadherin, an epithelial marker, significantly increased, whereas the expression levels of the mesenchymal marker, Vimentin, significantly decreased in 5637 and T24 cells following 24 h treatment with 60 or 80 µM celecoxib compared with the control group (P<0.05; Fig. 2B-D). Hence, the results of the present study indicated that celecoxib inhibits the proliferation, migration, invasion and EMT of BC cells.

EMT is thought to serve a key function in the invasion and metastasis of numerous tumor types (19). A body of literature has revealed that miRNAs have been implicated in the regulation of a variety of cellular functions and biological alterations including EMT (20-22). miR-145 is a regulator of EMT in numerous cancer types (23-26) including BC (27). It was revealed, in the present study, that the relative expression levels of miR-145 were significantly downregulated in BC cells compared with the control cells (P<0.05; Fig. 2E). Additionally, the levels of miR-145 in 5637 cells were higher compared with T24 cells. Notably, the expression of endogenous E-cadherin in 5637 cells has also been demonstrated to be greater compared with T24 cells (28-30). These results suggest that miR-145 may be a suppressor of EMT in BC cells. To investigate the molecular mechanism by which celecoxib reverses EMT in BC cells, the present study examined the levels of miR-145 in BC cells following treatment with celecoxib. Interestingly, as presented in Fig. 2F-G, celecoxib significantly increased the expression of miR-145 in 5637 and T24 cells in a time- and dose-dependent manners (P<0.05). This suggests that celecoxib may directly affect the transcriptional expression of miR-145, and not merely select for resistant populations that are inherently E-cadherin and miR-145 positive.

Initially, the effect of the transfection of miR-145 mimics and inhibitors in 5637 and T24 cells were confirmed using RT-qPCR (Fig. 3A). The present study then determined whether miR-145 was required for the celecoxib-mediated inhibition of EMT. The administration of celecoxib significantly inhibited the invasion ability of miR-NC inhibitor-transfected 5637 cells compared with the negative control group (P<0.05) but not the miR-145 inhibitor-transfected 5637 cells (Fig. 3B). In addition, the protein levels of E-cadherin were reduced and the Vimentin levels were increased in miR-145 inhibitor-transfected 5637 cells compared with the miR-NC inhibitor-transfected 5637 cells following the administration of celecoxib (Fig. 3C and D).

Tumor growth factor β (TGF-β) signaling have emerged as major inducers of EMT, particularly the TGF-β-induced Smad signaling pathway (31-34). miR-145 has been reported to affect TGF-β-induced EMT by directly targeting Smad3 and TGFBR2 (24,35,36). It was hypothesized that this may be the mechanism by which miR-145 mediates the inhibition of EMT induced by celecoxib. The putative targets of miR-145 were predicted by using the TargetScanHuman 7.2 website, and Smad3 and TGFBR2 were identified (Fig. 4A). In addition, the mRNA and protein levels of Smad3 and TGFBR2 in T24 cells were all significantly reduced following the administration of celecoxib and miR-145 overexpression (P<0.05; Fig. 4B and C).

A previous study demonstrated that celecoxib has potent anti-tumor effects in combination with BCG immunotherapy in an experimental model of murine BC (11). Another previous study revealed that the intravesical administration of exogenous miR-145 may inhibit tumor growth in mouse orthotopic human BC xenografts (37). However, to the best of our knowledge, no studies have previously reported the effect of the combination of celecoxib and miR-145 on BC. To understand the efficacy of the combined treatment of celecoxib and miR-145 mimic, the invasion and migration inhibitory effects of the combined treatment of celecoxib and miR-145 mimic on human BC were examined in vitro. The data indicated that celecoxib in addition to miR-145 mimics (20 nM) exhibited a significantly stronger anti-invasion and anti-migration ability compared with celecoxib alone (P<0.05; Fig. 5). Additionally, the expression levels of EMT associated proteins and miR-145 targeted proteins were measured using a western blot assay. The results indicated that celecoxib in combination with miR-145 mimic resulted in a significant increase in E-cadherin expression levels compared with celecoxib or miR-145 mimic alone (P<0.05; Fig. 6). In contrast, celecoxib and miR-145 mimic in combination resulted in a significant decrease in the expression levels of Vimentin, TGFBR2 and Smad3 compared with celecoxib or miR-145 mimic alone (P<0.05; Fig. 6).