We retrospectively reviewed the records of patients diagnosed with BC undergoing surgical resection at the First Affiliated Hospital of University of South China between January 2017 and January 2020. A total of 60 patients were enrolled as study subjects. None of these patients underwent preoperative chemoradiotherapy. Tumour tissues were collected, and adjacent normal tissue samples were used as controls. The clinical data and recurrence-free survival of these patients were retrospectively reviewed. The censor date for the recurrence-free survival data was set as a date 48 months after the surgery. The present study was approved by the Ethics Committee of the First Affiliated Hospital of University of South China (No. 20160308), and all patients signed the informed consent forms.
The human bladder cancer T24 and 5637 cell lines were purchased from the Cell Bank affiliated with Shanghai Institute of Biochemistry and Cell Biology. The cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% foetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were cultured in 5% CO2 at 37 °C.
PCR was used to amplify the full-length cDNA of human NEAT1. The primer sequences used in the present study were as follows: NEAT1: F-GGGGTACCCTTCCTCCCTTTAACTTATCCATTCAC and R-GGAAGCTTCATAACAACCATTACCACCTCCTTCTC. The ectopic vector pcDNA3.1-NEAT1 and the corresponding control vector were constructed. Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA) was used for transient transfection experiments according to the manufacturer’s instructions. The ectopic and control vectors were added to the cells of the NEAT1 and negative control (NC) groups, respectively. T24 and 5637 cells without transfection were used as the control (CON) group. The miR-101 mimic and inhibitor with the corresponding negative controls (miR-101 mimic, miR-101 inhibitor, miR-101 mimic NC and miR-101 inhibitor NC), the VEGF-C overexpression plasmid were purchased from RiboBio (Ribobio, Guangzhou, China).
Total RNA was isolated using TRIzol reagent (Invitrogen, Grand Island, NY, USA). Six microlitres of total RNA was subjected to reverse transcription by using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara, Shiga, Japan). QRT–PCR was performed using the StepOnePlus™ real-rime PCR system (Applied Biosystems, Foster City, CA, USA). The sequences of the PCR primers were as follows: NEAT1: F-CAGTTAGTTTATCAGTTCTCCCATCCA, R-GTTGTTGTCGTCACCTTTCAACTCT; miR-101: F-UACAGUACUGUGAUAACUGAA, R-CAGUUAUCACAGUACUGUAUU; U6: F-CTCGCTTCGGCAGCACA, R-AACGCTTCACGAATTTGCGT; VEGF-C: F-AGAAGGAGGAGGGCAGAAT, R-GTCTCGATTGGATGGCAGTAG; and GAPDH: F-GTGGTCTCCTCTGACTTCAAC, R-CCTGTTGCTGTAGCCAAATTC. The data were recalculated by the 2–ΔΔCt method after normalization against the corresponding endogenous controls (GAPDH and U6).
Western blot analysis
The cells were lysed in RIPA buffer and centrifuged at 12,000 rpm for 5 min. Then, the supernatant was collected. Protein concentrations were determined using a BCA protein assay (Beyotime Biotechnology, Shanghai, China). Thirty micrograms of protein was processed by electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Then, the membranes were blocked using skimmed milk for 1 h. After washing with TBST 3 times for 10 min each time, the membranes were incubated with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies. The proteins were visualized by enhanced chemiluminescence detection reagent (Thermo Scientific, Waltham, MA, USA). All data were normalized to GAPDH levels. Primary antibodies against VEGF-C (1:1000; ab9546, Abcam, USA) and GAPDH (1:1000; ab8245, Abcam, USA) were used. In the running of experiments, sectioning gels is performed in our research group, as the blots are cut prior to hybridisation with antibodies. So images showing full-length membranes were not provided, but all the replicate versions of original gel image were uploaded on supplemental files to confirm specific detection of the target antigen.
The cells were cultured in 96-well plates at a density of 5 × 104 cells per well. After overnight incubation, MTT reagent was added to each well, and the plate was incubated for 3 h at 37 °C. Then, the supernatant was removed, and 100 μL of DMSO was added to each well. Absorbance was recorded at a wavelength of 570 nm.
The cells at a density of 5 × 104 were transferred into the upper Transwell chamber containing serum-free medium. The lower Transwell chamber was filled with medium containing 10% serum. The cells were cultured for 8 h at room temperature. Migrated cells were fixed using 4% paraformaldehyde and stained with 0.1% crystal violet for 10 min. Cell migration was detected, and the number of migrating cells was calculated using an inverted microscope (Nikon, Japan,magnification × 200).
For the cell invasion assay, 5 × 104 transfected cells in 200 µL serum-free medium were added to the upper chamber of the Matrigel-precoated inserts (BD Bioscience, USA). The lower chamber was filled with medium supplemented with 10% FBS. After culture in 5% CO2 at 37 °C for 8 h, the cells invading into the lower chamber were stained with 0.1% crystal violet (Sigma, USA). PBS was used to wash the samples and remove excess stain. The number of the cells migrate through the membrane was counted using an inverted microscope (Nikon, Japan, magnification × 200).
For the luciferase reporter assay, the WT or MUT NEAT1 constructs were subcloned into the pGL3 basic vector (Promega, Madison, WI, USA). The cells were cotransfected with 5 µl miR-101 mimics and miR-101 NC and with 5 µg WT-NEAT1 or MUT-NEAT1, followed by incubation for 48 h. The luciferase activity was assayed using a Dual-Luciferase® reporter assay system (Promega, Madison, WI, USA), and firefly luciferase activity was normalized to the Renilla activity.
Orthotopic bladder cancer model
Female BALB/c-nu mice (4–6 weeks of age) were purchased from Nanjing Institute of Biomedicine (Nanjing, China; licence No.: SCXK(Su)2015-0001). Mice were randomly divided into 3 groups: the CON (n = 10), NC (n = 10) and NEAT1 groups (n = 10). After mice were anaesthetized successfully with 1% pentobarbital sodium (45 mg/kg), the lower abdomen of the mice was pressed to empty the bladder. A catheter (no. 7 venous indwelling needle) was inserted into the bladder and flushed with PBS. After treatment with 100 μl of 0.1 g/L HCl, 100 μL of 0.1 g/L KOH and phosphoric acid buffer, the external orifice of the urethra was ligated for 30 min. Then, Hank's balanced salt solution (HBSS) containing 1.5 × 106 cancer cells per 150 μL (T24/5637) was injected into the bladder and retained for 1 h. Four weeks after tumour cell inoculation, the mice were sacrificed to collect the tissues for subsequent experiments.
Haematoxylin and eosin staining
The tissue samples were fixed in 4% paraformaldehyde for more than 24 h and then embedded in paraffin. After cutting 4 μm-thick sections, the samples were subjected to routine steps of desiccation, haematoxylin–eosin staining, and clearing. The images were imaged under a microscope (magnification, × 200).
The tissue sections were blocked with 5% bovine serum albumin for 20 min and incubate with an anti-VEGF-C antibody (1:1000; ab9546, Abcam, USA) overnight at 4 °C. Then, the tissue samples were incubated with a secondary antibody at 37 °C for 30 min. The sections were then stained with DAB (R&D Systems, Minneapolis, MN, USA). The staining results and images were assessed using a light microscope (magnification, × 200).
Statistical analysis was performed using SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA). In vitro experiments were repeated in triplicate. The data are presented as the mean ± SD. The differences between the groups were determined using Student’s t test and one-way analysis of variance (ANOVA). One-way ANOVA followed by Tukey's post-hoc test was used to determine significance. Kaplan–Meier analysis with the log-rank test was used to evaluate the recurrence-free survival of patients. The p values less than 0.05 were considered statistically significant.