A total of 30 pairs of BC tissues and matched adjacent normal tissues (0.5 cm between BC tissues and normal tissues) were collected from patients with BC (40 to 75 years old, 17 male patients and 13 female patients who had not undergone chemotherapy before surgery) who received cystectomy between July 2018 and May 2019 at Sichuan Cancer Hospital & Institute (Chengdu, China). All samples were obtained after receiving informed consent from patients, and the study protocol was approved by the Ethics Committee of the University of Electronic Science and Technology of China [approval no. SYSK (Jing) 2018-0034]. The human BC cell lines J82 and T24, as well as the SV40 immortalized human uroepithelial cell line SV-HUC-1, were purchased from American Type Culture Collection, and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBC (HyClone; Cytiva) at 37°C in a humidified incubator containing 5% CO₂. These cell lines were verified via short tandem repeat profiling. Propofol was purchased from Shanghai Zhenrui Biotechnology Co., Ltd., and diluted in DMSO (Sigma-Aldrich; Merck KGaA) for in vitro assays.

Cell viability was evaluated using a CellTiter-Glo Luminescent Cell Viability Assay kit (cat. no. G7572; Promega Corporation) according to the manufacturer's instructions. Cells were treated with 10 µg/ml propofol or equal volume of DMSO as control for the cell viability assay. In brief, the cells and CellTiter-Glo reagents were kept at room temperature for 30 min, then mixed thoroughly and incubated in a dark room for 10 min. Then, the luminescence signal (400–700 nm) was recorded on a microplate reader (Multiskan SkyHigh; Thermo Fisher Scientific, Inc.). Each experiment was conducted in triplicate.

Total RNA from cell lines or tissue samples was extracted using TRIzol^® regent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, then reverse transcribed into cDNA using a PrimeScript™ RT kit (Takara Bio, Inc.), according to the manufacturer's protocol. qPCR was performed using SYBR Premix EX Taq™ (Takara Bio, Inc.). Reaction conditions for qPCR were as follows: 95°C for 1 min; then 40 cycles of 95°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec. The following primers were used: miR-145-5p forward, 5′-ACACTCCAGCTGGGAGTCT-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′; TOP2A forward, 5′-GGGAGAGTGATGACTTCCATATGGA-3′ and reverse, 5′-AACACCTTCCCCAAACTAAATTCAG-3′; U6 forward, 5′-CGCTTCGGCAGCACATATACTA-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCA-3′; and GAPDH forward, 5′-TGCACCACCAACTGCTTAGC-3′ and reverse, 5′-GGCATGGACTGTGGTCATGAG-3′. U6 and GAPDH were used to normalize the expression of miRNA and mRNA, respectively, and results were analyzed via the 2^−ΔΔCq method (23).

Total protein was isolated using RIPA lysis buffer (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) containing protease inhibitors (Beyotime Institute of Biotechnology). The protein concentration was determined using a BCA protein assay kit (Shanghai Zeye Biotechnology Co., Ltd.); then, 30 µg protein was separated via 10% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred onto PVDF membranes (Pall Life Sciences). The membranes were blocked with 5% skimmed milk at room temperature for 1 h, then incubated with the following primary antibodies at 4°C overnight: Anti-TOP2A (1:5,000; cat. no. ZY-6562-21R) and anti-GAPDH (1:2,000; cat. no. ZY-6909-37R; both Shanghai Zeye Biotechnology Co., Ltd.). The membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:4,000; cat. no. 98164; Cell Signaling Technology, Inc.) for 1 h at room temperature, then the hybridization signals were detected by Immobilon Western chemiluminescence (EMD Millipore) and captured using Amersham ImageQuant 600 imaging system (cat. no. 29083461; GE Healthcare). The semi-quantification of the western blots was conducted using ImageJ version 1.52 (National Institutes of Health).

The miR-145-5p antisense oligonucleotide inhibitor (anti-miR-145-5p, 5′-AGGGATTCCTGGGAAAACTGGAC-3′, anti-miR-NC, 5′-ACGGAGGCTAAGCGTCGCAA-3′) was purchased from Shanghai Shenggong Biology Engineering Technology Service, Ltd. TOP2A-specific small interfering (si)RNA was obtained from Santa Cruz Biotechnology, Inc., and the sequences were as follows: si-TOP2A, 5′-ACCTTTGACTCTCAGACAAAAGA-3′ and si-NC, 5′-CCAGTTATGCTGACATGTAT-3′. The expression plasmid for miR-145-5p was constructed by cloning the mature sequence (5′-GTCCAGTTTTCCCAGGAATCCCT-3′) into a pCMV-MIR lentiviral vector (cat. no. PCMVMIR; OriGene Technologies, Inc.). Empty pCMV-MIR lentiviral plasmid was used as an empty vector control (EV). Recombinant lentiviral particles were produced in 293T cells (American Type Culture Collection) via co-transfection with the helper plasmids pCMV–VSV-G, pRSV-REV and pMDL at a ratio of 5:1:5:5 using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). The virus-containing supernatant was harvested at 24, 48 and 72 h after transfection and filtered using 0.22-µm filters, then stored at −80°C for further use. For viral infection, 500 µl virus-containing supernatant was added to 1×10⁵ cells with 8 µg/ml polybrene overnight, then selected with puromycin (2 µg/ml) for 72 h. For transient transfection, cells (1×10⁶ cells) were seeded in 6-well plates and transfected with miR-145-5p mimics, anti-miR-145-5p or si-TOP2A using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. In brief, plasmids were mixed with Lipofectamine 3000 reagents and incubated with cells overnight, then used for further studies at 24 h post-transfection.

Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/gds/) was used to identify differentially expressed genes between BC samples and matched normal tissues in the GSE76211 dataset (24). RNA sequencing data was aligned using PRADA tool (https://www.rna-seqblog.com/prada-pipeline-for-rna-sequencing-data-analysis/). The RNA-seq reads were counted over gene exons using HTSeq V0.6.1 (https://htseq.readthedocs.io/en/release_0.11.1/). DESeq R package (1.10.1) (http://bioconductor.org/packages/release/bioc/html/DESeq.html) was used to evaluate the differences between the samples. P<0.05 and log2 fold-change ≥2 were used to select differentially expressed genes.

The targets of miR-145-5p were predicted using StarBase 2.0 (http://starbase.sysu.edu.cn/index.php). TargetScan 7.0 (http://www.targetscan.org) was used to identify potential binding sites between TOP2A and miR-145-5p. Cells (1×10⁶ cells) were seeded in 6-well plates; then, the wild-type (WT) 3′-untranslated region (3′UTR) of the TOP2A containing the putative miR-145-5p binding sites or a mutant (MUT) sequence was inserted into a pMIR plasmid containing a firefly luciferase reporter prior to transfection with Lipofectamine 3000. 293T cells were co-transfected with pMIR plasmid containing the 3′ UTR of WT TOP2A or MUT, miR-145-5p mimics or miR-NC, and a Renilla luciferase plasmid at a ratio of 2:2:1. After 48 h, the luciferase activity was detected using a dual-luciferase reporter assay system (Promega Corporation).

Cells were treated with 10 µg/ml propofol or equal volume of DMSO as a control for the wound-healing assay. In brief, cells (2×10⁵ cells) were seeded in a 12-well plate and cultured to full confluence. The cell monolayer was scratched across the center using a pipette tip and then washed three times with PBS to remove the detached cells. Cells were then cultured with 10% FBS (Cytiva; Hyclone) at 37°C in a humidified incubator containing 5% CO₂ (11). The wounds were observed with a microscope (Zeiss AG) at 0 and 24 h.

Cells were treated with 10 µg/ml propofol or equal volume of DMSO as a control for the Transwell invasion assay. In brief, cells (1×10⁴/well) were suspended in serum-free medium and seeded in the upper chamber with 8-µm pore filters (EMD Millipore). The filters were precoated with Matrigel (BD Biosciences) at room temperature for 24 h. The lower chamber was filled with complete medium. Following incubation for 24 h, invaded cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.5% crystal violet (Nanjing Jiancheng Bioengineering Institute) for 1 h at room temperature. The cells were observed with a microscope (Zeiss AG). For each sample, five fields of view were randomly selected.

The animal study was approved by the Ethics Committee of the University of Electronic Science and Technology of China. Six-week old BABL/C nude mice (25–30 g) were housed in individually ventilated cages under specific pathogen-free conditions at 25°C and 12 h light/dark cycle. Mice were allowed access to sterilized water and food ad libitum. First, T24 cells in the logarithmic phase were transfected with anti-miR-145-5p oligonucleotide inhibitor or anti-miR-negative control (NC), then injected subcutaneously into 15 female BALB/C nude mice (Shanghai Model Organisms Center, Inc.) 24 h post-transfection. Mice were randomly divided into 3 groups (n=5/group): Vehicle + anti-miR-NC group; propofol + anti-miR-NC group; and propofol + anti-miR-145-5p group. The propofol + anti-miR-145-5p group was injected with T24 cells transfected with anti-miR-145-5p, then intraperitoneally injected with 10 mg/kg propofol. The propofol + anti-miR-NC group was injected with T24 cells transfected with anti-miR-145-5p, then treated with an equal volume of vehicle control (Soybean oil). The vehicle + anti-miR-NC group was injected with T24 cells transfected with anti-miR-NC, then treated with an equal volume of vehicle control. Propofol was intraperitoneally injected daily for 4 weeks. Tumor growth was measured via a two-dimensional measurement method and calculated as follows: Volume=(A²xB)/2, where A was the smallest diameter of the tumor and B was the largest diameter of the tumor. All mice were anesthetized via inhalation of 3% isoflurane and sacrificed via cervical dislocation at the end of the experiment.

Mouse xenograft samples were fixed in 10% formalin at room temperature overnight, embedded in paraffin and cut into 3-µm sections. Endogenous peroxidase activity was blocked with 3% H₂O₂ at room temperature for 15 min. Antigen retrieval was performed in citrate buffer and sections were washed with PBS. Then, sections were subsequently blocked with 1% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Next, the sections were incubated with primary antibodies against E-cadherin (1:200; cat. no. 3195; Cell Signaling Technology, Inc.) or vimentin (1:200; cat. no. ab92547; Abcam) at 4°C overnight and the biotin-conjugated secondary antibody (1:3,000; cat. no. 14708; Cell Signaling Technology, Inc.) at room temperature for 10 min. Streptavidin-peroxidase was applied for 15 min at room temperature followed by development with DAB. Then, the sections were counterstained with Mayer's Hematoxylin for 2 min at room temperature. Subsequently, the sections were dehydrated and sealed with neutral gum. Images were photographed using the LSM 5 Pa Laser Scanning Microscope (Zeiss AG).

SPSS 20.0 software (IBM Corp.) was used to perform statistical analysis. All experiments were repeated at least three times and data were presented as the mean ± SD. Comparisons between two different groups were performed using independent-samples t-tests or paired t-tests, and comparisons of multiple groups were performed using one-way ANOVA with Bonferroni's post hoc test. The correlation between miR-145-5p and TOP2A mRNA was evaluated using Spearman's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

The expression of miR-145-5p in BC samples and cell lines was measured via RT-qPCR. Compared with paired normal tissues, miR-145-5p expression was significantly downregulated in BC tissues (Fig. 1A). Similarly, miR-145-5p was downregulated in human BC cell lines (J82 and T24) compared with the human uroepithelial SV-HUC-1 cell line (Fig. 1B). To explore the effects of propofol on miR-145-5p expression, J82 and T24 cells were treated with propofol and evaluated for miR-145-5p expression at different time points. It was found that miR-145-5p expression was induced by propofol in both J82 and T24 cells in a time-dependent manner (Fig. 1C and D). J82 and T24 cells were exposed to increasing concentrations of propofol (0–20 µg/ml) for 24, 48 and 72 h, then cell viability was evaluated. It was found that propofol reduced the viability of BC cells in a dose-dependent manner (Fig. 1E and F). Collectively, these findings indicated that propofol induced miR-145-5p expression in BC cells.

To study the effects of propofol in BC cells and whether its biological effects were associated with miR-145-5p expression, J82 and T24 cells were transfected with miR-145-5p lentivirus or anti-miR-145-5p oligonucleotides, then exposed to propofol for cell viability, wound-healing and Transwell cell invasion assays. The expression of miR-145-5p in J82 and T24 cells was significantly downregulated via transfection with anti-miR-145-5p and upregulated via transfection with miR-145-5p, compared with the respective negative controls (Fig. 2A). Then, J82 and T24 cells transfected with miR-145-5p or anti-miR-145-5p were exposed to propofol, and the expression of miR-145-5p was evaluated via RT-qPCR. It was observed that propofol-induced miR-145-5p expression in J82 and T24 cells was attenuated by transduction with anti-miR-145-5p (Fig. 2B and C). In a cell viability assay, it was observed that propofol treatment or miR-145-5p overexpression decreased the viability of J82 and T24 cells, while anti-miR-145-5p reversed the effects induced by propofol treatment (Fig. 2D and E). Wound-healing assays demonstrated that cell migration was significantly reduced when J82 or T24 cells were treated with propofol or transduced with miR-145-5p compared with the vehicle control group (equal volume of DMSO). As expected, the extent of wound closure was significantly increased in the propofol + anti-miR-145-5p group compared with the propofol + anti-miR-NC group (Fig. 2F and G), indicating that propofol reduced the migration of BC cells by promoting miR-145-5p expression. In Transwell cell invasion assays, propofol treatment and miR-145-5p overexpression significantly decreased the number of invading cells compared with the control group, while depletion of miR-145-5p via transduction with anti-miR-145-5p attenuated the effects induced by propofol treatment (Fig. S1A and B). Collectively, the results indicated that propofol suppressed the proliferation, migration and invasion of BC cells, and that this effect was partially mediated by regulating miR-145-5p expression.

The top 10 upregulated and downregulated mRNA molecules in the GSE76211 from the GEO were screened and are shown in a heat map (Fig. 3A). TOP2A (log₂ fold-change=4.5; P<0.001) was the most affected mRNA among the 20 targeted mRNAs; thus, it was hypothesized that TOP2A may be a direct target of miR-145-5p. TargetScan 7.0 was used to identify potential binding sites between TOP2A and miR-145-5p (Fig. 3B). To reveal the direct effect of miR-145-5p on TOP2A, miR-145-5p was overexpressed in J82 and T24 cells, and TOP2A expression was evaluated. It was found that the mRNA expression of TOP2A was significantly downregulated following miR-145-5p overexpression (Fig. 3C). In luciferase reporter assays, the luciferase activity was significantly reduced by co-transfection of miR-145-5p + TOP2A 3′UTR-WT compared with the control, whereas the luciferase activity in cells co-transfected with miR-145-5p + TOP2A 3′UTR-MUT was not significantly altered (Fig. 3D and E). The expression of TOP2A was evaluated in BC samples and paired normal tissues, and it was found that both the mRNA (Fig. 4A) and protein expression levels (Fig. 4B) of TOP2A were significantly upregulated in tumor samples compared with paired normal tissues. Consistent with this, it was revealed that TOP2A mRNA (Fig. 4C) and protein (Fig. 4D) expression was increased in J82 and T24 cells compared with SV-HUC-1 cells. In addition, it was observed that when treated with propofol for 72 h, the mRNA (Fig. 4E) and protein (Fig. 4F) levels of TOP2A were significantly reduced compared with the control, indicating that propofol inhibited the expression of TOP2A. Furthermore, correlation analysis of miR-145-5p and TOP2A mRNA expression in BC samples revealed that miR-145-5p and TOP2A were significantly negatively correlated (Ρ=−0.835; P=0.0011; Fig. 4G). Collectively, these results indicated that TOP2A was directly targeted by miR-145-5p in BC cells.

To investigate whether the antitumor effects of propofol were associated with the miR-145-5p/TOP2A axis, TOP2A expression was knocked by transfecting J82 and T24 cells with anti-miR-145-5p or si-TOP2A. It was demonstrated that TOP2A expression was increased in J82 and T24 cells transduced with anti-miR-145-5p compared with anti-miR-NC (Fig. 5A), while cells transfected with si-TOP2A exhibited downregulated expression of TOP2A compared with si-NC (Fig. 5B). Then, cells were transfected with anti-miR-NC, anti-miR-15-5p, si-NC or si-TOP2A as indicated, and then exposed to propofol. It was revealed that the mRNA (Fig. 5C and D) or protein expression levels (Fig. 5E and F) of TOP2A in J82 and T24 cells were suppressed by propofol treatment, and that this effect in propofol-treated cells was attenuated by anti-miR-145-5p, which was again reversed by si-TOP2A transfection, compared with the corresponding negative controls. In wound-healing (Fig. S2A and B) and Transwell assays (Fig. S2C and D), propofol suppressed the migration and invasion of J82 and T24 cells, which was partially attenuated by anti-miR-145-5p and further exacerbated by subsequent TOP2A knockdown. Taken together, these results indicated that propofol exerted antitumor effects via the miR-145-5p/TOP2A axis.

In order to further verify the antitumor effect of propofol in vivo, a BC cell xenograft nude mouse model was constructed. T24 cells were transfected with anti-miR-145-5p lentiviral vectors or anti-miR-NC, and then injected into female BALB/C nude mice prior to treatment with 10 mg/kg propofol for 4 weeks. It was revealed that tumor volume and weight were significantly decreased in the propofol + anti-miR-NC group compared with the vehicle + anti-miR-NC group, whereas this effect was attenuated in the propofol + anti-miR-145-5p group, indicating that propofol suppressed the proliferation of BC cells, which was partially attenuated by miR-145-5p knockdown (Fig. 6A-C). Moreover, the expression of miR-145-5p was upregulated by propofol and downregulated by anti-miR-145-5p in tumor tissues (Fig. 6D). Conversely, the mRNA and protein expression levels of TOP2A were suppressed by propofol; this effect was attenuated by anti-miR-145-5p (Fig. 6E and F). The expression of E-cadherin and vimentin was investigated in tumor xenograft tissues from the different groups, and it was revealed that E-cadherin expression was increased, while that of vimentin was downregulated, by propofol, and that these effects were attenuated by anti-miR-145-5p (Fig. 6G and H). Collectively, these findings indicated that propofol suppressed tumor growth by regulating the miR-145-5p/TOP2A axis in vivo.