Patient samples

The research protocol was reviewed by the Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University (approval no.: 2020–436). Informed consents were received from all participants. 39 matched bladder cancer and para-carcinoma specimens were obtained from patients who underwent operation in the First Affiliated Hospital of Jinzhou Medical University (Jinzhou, China) from March 2019 to June 2021. No patients had been treated with pre-operative anti-tumor therapies. The age of the patients were ranged from 42 to 84 year-old. Additionally, metastasis was found in 21 participants. Then the samples were kept at -80 °C. The inclusion criteria were as followed: 1) Able to provide written in formed consent; 2) Documentary evidence of a pathologically confirmed primary bladder cancer; 3) Consent to access archival tumor material; 4) Ascertainment of demographic data, height and weight, drug history and family history of bladder cancer and other cancer. The exclusion criteria were as followed: 1) Inability to provide informed consent; 2) history of any other primary malignancy except bladder cancer; 3) patient underwent adjuvant treatment such as radio- and chemo-therapy.

Cells

SV-HUC-1 and human bladder cancer cells (T24, 5637, J82 and RT4) were obtained from the Institute of Cell Biology (Shanghai, China). Cells were cultured using DMEM (Sigma, St. Louis, MO, USA) containing FBS (10%), penicillin (100 U/ml) and streptomycin (100 µg/ml; GE Healthcare Life Science) and incubated at 37℃ with 5% CO2.

Transfections

Vectors that overexpress FZD8 (oe-FZD8) together with the control oe-NC, miR-99a-5p mimic together with the control miR-NC, and small interfering RNA targeting circMCTP2 (sh-circMCTP2) together with the control sh-NC were purchased from Gene Pharma (Shanghai, China). T24 and RT4 cells were inoculated and cultured using DMEM containing no antibiotics. ~ 25 nM of vectors were utilized for transfection. All the transfections were conducted using Lipofectamine 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Twelve hour following transfection, culture media was replaced with DMEM with 10% FBS. Twenty-four hours following the transfection with sh-circMCTP2, cells were treated with GSK-3b inhibitor (SB216763, Enzo Life Sciences, Farmingdale, NY, USA) at a concentration of 10 µM. After twelve hours of treatment, cell viability, migration, invasion and apoptosis were evaluated.

Rever transtription-quantitative polymerase chain reaction

TRIzol (Sobao Biotechnology, Shanghai, China) was utilized for the extraction of RNA from clinical specimens or T24 and RT4 cells. Briefly, cDNA was generated using PrimeScript™ RT (Invitrogen, Shanghai, China). Then, qPCR was conducted by ABI 7500 (Thermo Fisher Scientific, Inc.). The program used for qPCR were: 95℃ for 5 min, then 45 cycles of 95℃ for 15 s 60℃ for 30 s and 72˚C for 10 s. The expression of genes was evaluated using 2−ΔΔCT method. The forward and reverse primers were: miR-99a-5p: forward, 5’-TGGCATAAACCCGTAGATCC-3’ and reverse, 5’-CCATAGAAGCGAGCTTGTG-3’; circMCTP2: forward, 5’- ACCAGAAGAGCCAGAGGAGTC-3’, and reverse, 5’-TGGCCTGGTCCGCTGTTTTAA-3’; FZD8: forward, 5’- GGACTACAACCGCACCGACCT-3’ and reverse, 5’- ACCACAGGCCGATCCAGAAGAC-3’; GAPDH: forward, 5’-CGCTCTCTG CTCCTCCTGTTC-3’ and reverse, 5’-ATCCGTTGACTCCGACCTTCAC-3’; U6: forward, 5’-GTAGATACTGCAGTACG-3’ and reverse, 5’-ATCGCATGACGTACCTGAGC-3’.

Dual luciferase reporter assay

Dual luciferase reporter plasmids (circMCTP2-WT, circMCTP2-MUT, FZD8-WT, FZD8-MUT) were generated by Promega (Promega, USA). Liposome 2000 (Invitrogen, Arsbad, CA, USA) was utilized for co-treatment of 293 T cells with circMCTP2-WT, circMCTP2-MUT, FZD8-WT, FZD8-MUT and miR-99a-5p mimics or miR-NC. The activity of luciferase was examined 48 h following treatment.

Western blotting

The lysis of T24 and RT4 cells was performed using RIPA buffer (Thermo Fisher Scientific). Then protein samples were separated by 10% SDS-PAGE and then transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were further blocked using 5% skimmed milk for 1 h and incubated with primary antibodies against FZD8 (1:1000, cat. no. ab155093, Abcam, MA, USA), b-catenin (1:2000, cat. no. ab224803, Abcam), cyclin D1 (1:2000, cat. no. ab32053, Abcam) or GAPDH (1:2000, cat. no. ab9485, Abcam) in cold room overnight. The following day, membranes were incubated with HRP-conjugated secondary antibody (1:3000, cat. no. 7074, Cell Signaling Technology). The blots were visualized by ECL kit (Pierce Biotechnology; Thermo Fisher Scientific) and quantified using Quantity One software.

CCK-8 assay

Briefly, T24 and RT4 cells were seeded on 96 wells plates. At 24, 48, 72 and 96 h post-treatment, 10 μl of CCK-8 solution (CCK-8; Dojindo Molecular Technologies, Inc., Japan) was aliquoted into each well. Then the cells were kept in the incubator for 2 h and the absorbance (wavelength = 450 nm) was recorded using microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Assessment of cell migration and invasion

Chambers precoated with Matrigel (pore size = 8 μm; ChenGong Biotechnology, Shanghai, China) was utilized to assess the invasive activity only. In brief, 5 × 104 of treated cells were seeded onto the upper chamber, and 500ul of culture media was added to lower compartment. Twenty four hours later, cells left in the upper compartment were discarded. Then fixation of membrane was performed using 4% paraformaldehyde (10 min). Subsenquently, cells stained using crystal violet (0.5%) were counted using microscope.

Evaluation of cell apoptosis

Apoptosis assay (Baiying Biotechnology, Guangzhou, China) was utilized to evaluate the apoptosis of T24 and RT4 cells. Cells were washed and well mixed with PBS. For the evaluation of apoptosis, cells were well suspended and staining with PI (10 µg/ml) and annexin V-FITC (50 µg/ml) was carried out. Cells were kept at 4˚C for half an hour in dark. Subsequently, the apoptotic rate of cells was determined using a cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the results were interpreted by CellQuest analysis software (BectonDickinson, USA).

In vivo study

T24 cells transfected with sh-circMCTP2 or sh-NC were used for inoculation into nude mice. BALB/C nude mice were purchased from the Laboratory Animal Research Centre of Jinzhou Medical University. Briefly, the mice were housed under a temperature-controlled condition (22 ± 2˚C) with ~ 60% humidity, under a 12-h dark/light cycle with libitum access to food and water for at least three days before the experiments. Mice were randomly grouped (n = 5 in each group) and injected with T24 cells. A total of 1 × 107 cells were diluted in 200 μl PBS and injected into the back subcutaneously. Mice were then monitored four times a week. 42 days following the injection, mice were sacrificed and the tumor tissues were evaluated. Tumor volume was calculated using the following formula: V (mm3) = (length x width2)/2.

Statistical analysis

SPSS statistical software (version 17.0, SPSS Inc., Chicago, IL, USA) was utilized to analyze the data. Data were shown as mean ± standard error of mean (SEM). Difference between two comparison groups was interpreted using t-test, and that among more than two groups was interpreted by one-way ANOVA and post-hoc Tukey test. To evaluate the association of variables, Pearson’s correlation analysis was carried out. The survival rate was determined using Kaplan–Meier analysis, and log-rank method was conducted to investigate the difference of groups with high- and low-circMCTP2-expression. P < 0.05 was statistically significant.