Bioinformatic analysis
GEPIA (http://gepia.cancer-pku.cn), an online gene expression profiling interactive analysis tool, was used to analyze the expression of TRPM2-AS and GINS2 mRNA in tumor and normal bladder tissues. GSE37815 data series downloaded from GEO database provided the mRNA expression profile in bladder cancer. ENCORI (http://starbase.sysu.edu.cn/panCancer.php) was used to predict the miRNAs that could bind to TRPM2-AS, and TargetScan Human v7.2 algorithm was used to predict the target regulatory miRNAs that could bind to the 3’UTR of GINS2 mRNA.
Tissue samples collection
We collected BLCA tissues and corresponding adjacent tissues of 38 patients who were diagnosed with BLCA in The First Affiliated Hospital of Zhengzhou University hospital during the last three years. The processing of clinical tissue samples is in strict compliance with the ethical standards of the Declaration of Helsinki, and the ethics committee of the authors’ institutes. All the patients signed the informed content, and their clinical characteristics were summarized in Table 1.
qPCR
Total miRNA was dissociated using miRcute miRNA Isolation Kit (DP501, Tiangen Biochemical, China) based on the manufacture protocol with checking the purity of the RNA by using gel electrophoresis. MiR-22-3p reverse transcription was conducted using miRcute Plus miRNA First-Strand cDNA Kit (KR211, Tiangen Biochemical, China), while TRPM2-AS and GINS2 reverse transcription was carried out following the instruction of PrimeScript RT reagent Kit (Takara, RR037A, Japan). After reverse transcription, quantitative PCR was conducted using TB Green Premix Ex Taq II (Takara, RR820A, Japan). 2−ΔΔCt method was used to calculate RNA levels.We used GAPDH as the reference gene for TRPM2-AS and GINS2 mRNA, and U6 for miR-22-3p. All the primers were provided by GeneCopoeia (Guangzhou, China), and their sequences were provided in Table 2.
Cell line acquisition and cell culture
Both cancerous and normal cell lines were used in this study. The human BLCA cell lines RT4, T24, J82 and 5637, and normal urothelial epithelial cell line SV-HUC-1 were all acquired from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. They were cultured in RPMI 1640 media with 10% serum in a humidified incubator with 5% CO2 at 37℃.
Subcellular fractionation location
Cytoplasmic and nuclear RNA of T24 and 5637 cells was separated by Invitrogen PARIS Kit (ThermoFisher, AM1921, USA) following specifications with GAPDH serving as a cytoplasmic control and U2 as a nuclear control. The expression levels of TRPM2-AS, GAPDH, and U2 were measured using qPCR. Each experimental procedure was processed at least three times.
Cell transfection
Negative control (NC) plasmids, miR-22-3p inhibitor, and small interfering RNAs for TRPM2-AS (si-TRPM2-AS) and GINS2 (si-GINS2) were acquired from GeneCopoeia (Guangzhou, China). The transfection of these molecules into T24 and 5637 cells (plated in 6-well plates, 3x104/mL) was conducted using Lipofectamine 2000 reagent (Cat#: 11668027, ThermoFisher, USA). Then transfection lasted 48 hours.
Cell viability assay
Cell viability was detected using WST-8 method. Briefly, 4,800 transfected T24 and 5637 cells were seeded into 96-well plates, and grown for 24, 48, 72 hours, respectively. The 10μL cell counting kit-8 reagent (MedChemExpress, HY-K0301, China) was added to the cells for 4 h in the cell culture incubator. Lastly, the optical absorbance of every well was read at 450 nm under a microplate reader.
BrdU incorporation ELISA assay
BrdU DNA incorporation assay was used to evaluate cell proliferation ability. 3×104/mL T24 and 5637 cells were seeded into 96-well plates to grow for a day, and 10μL BrdU (5-bromo-2'-deoxyuridine, a thymidine analog, Abcam, ab126556, UK) was added to each well for 4 h. Thereafter, the fixing solution was added to each well to denature the cellular DNA. Then, every well was aspirated and washed. 100 μL/well anti-BrdU primary antibody was added to every well to allow a 1 h reaction. Every well was aspirated and washed again, and 100 μL/well Peroxidase conjugate Goat Anti-Mouse IgG secondary was added to incubate for 0.5 h. 100 µL/well TMB Peroxidase substrate was added and incubated for 0.5 h in the dark. The BrdU incorporated cells will be visible in blue, and the intensity of the blue color represents the proliferating abilities. To further quantify the proliferation, 100 µL of Stop Solution was added to each well, and the optical absorbance of each well was read at 450 nm under a spectrophotometric microtiter plate reader. All procedures were conducted at room temperature, and experiments in every group were repeated for at least three times.
Flow cytometry assay
Flow cytometry was an important method to measure cell death and identify the early and late apoptotic cells. Briefly, 1×105/mL T24 and 5637 cells in every group were rinsed in PBS for three times, and fixed in cold methanol for 30 min at 4 °C. Subsequently, the cells were suspended in 100 μl 1×binding buffer. PBS was subsequently added to each well to wash the cell suspension for three times. Then, the cells were incubated with 5 μL Annexin V-FITC and PI staining solution for 20 min in the dark. The cells then went through the FACS scan flow cytometer (Cytoflex, Beckman Coulter) within the next one hour. Both early (Annexin V positive and PI negative populations) and late apoptosis (Annexin V positive and PI positive staining) rate were counted as the apoptosis rate in this study.
Caspase-3 activation assay
Caspase-3 activation is an essential process during cell apoptosis. This assay was conducted in our study to further investigate into the cell apoptosis conditions in every group. The caspase-3 activation was assessed using a Colorimetric Caspase-3 Assay Kit (Cat#: ab39401, Abcam, UK). Briefly, 1×106 transfected T24 and 5637 cells were suspended in 50 μL cold cell lysis buffer for 10 minutes. The protein concentration was detected and adjusted to ~150 μg protein /50 μL reaction buffer. Then, 50 μL reaction buffer containing 10 mM DTT and 5 μL 4 mM DEVD-p-NA substrate was added to the cell lysates for an hour. Lastly, the optical absorbance was read at 405 nm using a spectrometer.
Luciferase reporter assay
The plasmids containing the wild strains and mutants of TRPM2-AS and GINS2 mRNA that contain the predicted binding sequences were constructed by and purchased from GeneCopoeia (China). These plasmids were transfected into the T24 and 5637 cells using Lipofectamine 2000 reagent (Cat#: ThermoFisher, 11668027, USA). Then, miR-22-3p mimic or NC were co-transfected into the transfected T24 and 5637 cells using Lipofectamine 2000 reagent. 48 h later, the cells were gathered to be lysed using lysis buffer. The dual-luciferase reporter assay system (Cat#: LF031, GeneCopoeia, China) was used to analyze the relative luciferase activity.
RNA immunoprecipitation (RIP)
RIP immunoprecipitation assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, 17-700, USA). Briefly, the BLCA cells were lysed in RIP lysis buffer including magnetic beads conjugated with anti-Argonaute2 (AGO2) or anti-IgG antibodies. Samples were incubated with Proteinase K and the immunoprecipitated RNA was isolated with TRIzol Reagent (ThermoFisher, 15596026, USA). Finally, the enrichment of TRPM2-AS and miR-22-3p was measured by qRT-PCR.
RNA pull-down assay
The 5×106 T24 and 5637 cells were treated with 0.5 mL of 25 mM Tris-HCl, 0.05% NP-40, 70 mM KCl, 2.5 mM EDTA, 80 U/mL RNase inhibitor and 1×protease inhibitor cocktail on ice for 20 minutes. After centrifugation, the supernatant was collected and incubated with biotin-coupled miR-22-3p (bio-miR-22-3p) or biotin-coupled NC (bio-NC) for 2 hours. After incubation, the cell lysate containing bio-miR-22-3p or bio-NC was incubated with streptavidin magnetic beads. After 4 hours, the beads were washed with reaction buffer to isolate the bound RNAs in the pull-down complex, and the expression of pull-down RNA was detected by qRT-PCR.
Western blot assay
The protein of cells was extracted using RIPA Lysis Buffer containing 5 mM EDTA and PMSF (P0013B, Beyotime, China). Different groups of proteins were quantified by a spectrophotometer so that the proteins were at the same level. 20μg of total protein in these groups was separated by 10% SDS-PAGE gel. Total protein was transferred onto PVDF membranes (Millipore, MA, USA) with 5.0% evaporated milk handled for 1 hour at 37 °C. Next, PVDF membranes were incubated with the primary antibodies against GINS2 (1:500, Cat#: ab197123, Abcam, UK) and GAPDH (1:5000, Cat#: ab175781, Abcam, UK) at 4°C overnight. Next day, the PVDF membranes were washed with PBS three times and incubated with Goat Anti-Rabbit IgG H&L secondary antibody (1:5000, Cat#: ab205718, Abcam, UK) at room temperature for 1 hour. ECL Substrate Kit (Cat#: ab133406, Abcam, UK) was used to strengthen the band signals. FluorChem FC2 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to read the intensities of the protein bands.
Statistical analyses
We analyzed our results using the Graphpad Prism v7.0 software. Data came from at least three independent results of every experiment, and were presented as mean±SD. The Wilcoxon test method was used to identify the significance of the differences between two groups, while one-way ANOVA with Dunnett’s post hoc method was used to identify the significance of the differences among multiple groups. 0.05 was the cut-off value for statistical significance.