Human tissue sample preparation
Bladder cancer tissue and the corresponding adjacent normal tissue were obtained from a total of 60 BC patients who received the same cisplatin-based chemotherapy (two cycles of adjuvant chemotherapy with cisplatin and gemcitabine after radical cystectomy) at Shengjing Hospital of China Medical University from 2016 Apr 5th to 2019 Jun 1st. This study gained the approval of the Ethics Committee of Shengjing Hospital of China Medical University, and a written informed consent was signed by each patient. Besides, the blood samples from above 60 BC patients were collected ahead of surgery, and 30 sex- and age-matched healthy individuals as the control group. All tissue samples were placed immediately into liquid nitrogen and stored at -80 ℃. The serum samples were extracted from the collected blood samples following the standard protocols. The clinicopathological features of BC patients enrolled in this research are listed in Table 1.
Human bladder cancer cell lines (BIU-87 and T24) and immortalized cells of normal human bladder epithelium (SV-HUC-1) were obtained from American Type Culture Collection and cultured in 1640 medium containing 10% FBS and 100 U/ml penicillin and streptomycin (Sigma, USA). Cells were cultivated at 37℃ in a humidified CO2 (5%) atmosphere. The chemosensitive BIU-87 and T24 cells were subjected to increasing doses of cisplatin in a stepwise way to establish cisplatin-resistant BC cell lines BIU-87R and T24R. The drug-resistant phenotype of BIU-87R and T24R were retained by maintaining the surviving cells in conditioned medium supplemented with 1 μg/mL cisplatin (Sigma).
Quantitative real-time PCR (qRT-PCR) assay
Total RNA was extracted from cells, tissues or serums using an RNAiso Plus kit (TaKaRa, Japan) according to manufacturer’s protocol. Quantitative real-time PCR assay was conducted to assess the expression of circCEP128 using SYBR green kit (TaKaRa, Japan) on the Light Cycler 480 (Roche, Switzerland) according to the manufacturer's recommendations. The primers were synthesized by Shanghai Sangon Biotech (China). GAPDH was used as an internal control. Relative gene expression levels were calculated using the 2-△△CT method. The primers used are listed as follows: circCEP128 forward primers: 5′-ACCCACATCGCTGGTTAGC-3′, reverse primers: 5′-TCGATCACCTTCTGCTTTCGT-3′; GAPDH forward primers: 5′- GGAAAGCTGTGGCGTGAT-3′, reverse primers: 5′-AAGGTGGAAGAATGGGAGTT-3′.
The siRNAs for circCEP128 and NC were ordered from Genepharm (Shanghai, China) and transfected into BIU-87R or T24R cells using Lipofectamine 3000 in accordance with the instructions. 48h after transfection, cells were harvested for further experiments. Expression level of circCEP128 was evaluated by qRT-PCR. Three siRNAs were designed with the following sequences for circCEP128: si-circCEP128-1: 5′-CUGUCAGCUGCAUGGAGCUUCGU-3′, si-circCEP128-2: 5′-GAGAGCUUGAACAGGAAUU-3′, si-circCEP128-3: 5′-GCACTGAGCCATTGTGAAT-3′ (si-circCEP128-2 was demonstrated to be the most effective inhibitory sequence and si-circCEP128 mentioned in the following results refers to si-circCEP128-2) and the corresponding si-NC sequence was 5′-AAUUCUCCGAACGUGUCACGU-3′.
BIU-87R or T24R cells were collected and inoculated in 96-well plates (1000 cells per well) 24h before transfection. Cells were cultured in 1640 medium supplemented with different concentrations of DDP (0, 1, 2, 4, 8, 16μg/mL) 48h after transfection of siRNAs in 96-well plates, with three replicate wells for each concentration. Cell viability was detected using Cell Counting Kit-8 (Beyotime, China) after 48h of incubation, based upon provided directions. A microplate reader was employed to determine the absorbance at 450 nm for each well.
Cell colony formation assay
BIU-87R or T24R cells transfected with siRNAs were collected and plated in 6-well plates (200 cells per well), and then cultured in medium supplemented with 3μg/mL cisplatin at 37 °C for 2 weeks. Afterwards, cells were fixed and subjected to 0.1% staining of crystal violet, the numbers of colonies were then counted for each well. Triplicate independent experiments were conducted.
Western blot analysis
The standard procedures were followed in the western blot analysis. In brief, the isolated protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. Then the separated proteins were blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA) by electrotransfer. The membrane was then blotted with primary antibodies against PCNA, Cyclin D1, RIPK3, p-RIPK3, MLKL or p-MLKL (1:1000, Abcam, UK), followed by horseradish peroxidase (HRP)-labeled secondary antibody (BIBC, China) incubation. All bands were detected by an ECL Western blot kit (Thermo Fisher Scientific, USA) and analyzed by Lab Works (TM ver4.6, UVP, Bio Imaging Systems, NY, USA). GAPDH was used as control.
Statistical analysis was conducted using SPSS 22.0 software (IBM) and Graph pad Prism 5.0. Differences between BC tissues and its corresponding adjacent normal tissues were analyzed using the Student’s t test, one-way ANOVA was used to evaluate the significance of differences between circRNA expression levels and clinicopathological features. The Kaplan-Meier survival analysis was performed to establish the overall survival. A receiver operating characteristic (ROC) curve was generated to assess its diagnostic value. p values < 0.05 were considered statistically significant.