Data preparation and processing
We downloaded primitive data (expressing profiles) and clinic information of human Bladder Urothelial Carcinoma (BLCA) from TCGA database (https://portal.gdc.cancer.gov/). Available mRNA sequencing (mRNA-seq) data and miRNA sequencing (miRNA-seq) data from 408 BLCA samples were gained from TCGA database. All raw RNA-seq data (miRNAs and mRNAs) were normalized as fragments per kilobase of exon model per million mapped fragment reads. Transformation of miRNA sequences into human mature miRNA names was accomplished using the miRBase database (https://www.mirbase.org/).
Screening of differentially expressed RNAs (DERNAs)
When performing the differential expression analysis in OCT4Bhigh and OCT4Blow BLCA samples, we determined the DERNAs (including lncRNAs, miRNAs, and mRNAs) with thresholds of |logFC| >2 and p <0.05. Volcano plots of the DERNAs [including differentially expressed lncRNAs (DElncRNAs), differentially expressed miRNAs (DEmiRNAs), and differentially expressed mRNAs (DEmRNAs)] were visualized using R language (version 4.1.2).
Establishment of the ceRNA network in BC
The ceRNA network was constructed by the following steps: (1) StarBase (http://starbase.sysu.edu.cn/) and TargetScan (version 7.2, http://www.targetscan.org/) was used to predict the DEmiRNAs and DEmRNAs, and build the miRNA-mRNA interaction pairs; (2) StarBase database was used to forecast the DEmiRNAs and DElncRNAs, and build the lncRNA-miRNA interaction pairs; (3) the VennDiagram package in R software was utilized to compare the target genes with DEmRNAs, and the target genes that overlapped with DEmRNAs in this study were selected for the next analysis to build the lncRNA-miRNA-mRNA triple regulatory network.
The Cytoscape plug-in cytoHubba was performed to identify the hub lncRNA-miRNA-mRNA triple regulatory network. The generated networks were visualized by Cytoscape software (version 3.7.0, https://www.cytoscape.org/).
Functional enrichment analysis
For the sake of understanding the possible biological processes and pathways of the network, we firstly conducted a functional enrichment analysis of the DERNAs in the lncRNA-miRNA-mRNA triple regulatory network in Metascape (http://metascape.org/gp/index.html). Then, GO enrichment (including BP, CC, and MF) and KEGG pathway analyses of these RNAs were performed using Metascape.
Survival analysis and construction of a specific prognosis model for BC
The survival status and time of BLCA patients were gained from TCGA clinical dataset. We used R software to perform Kaplan-Meier analysis and a log-rank test to determine the relationship between the OCT4-pg5/miR-145-5p/OCT4B ceRNA network with the overall survival (OS) of BLCA patients in TCGA database.
Cell culture and clinical samples
Five human bladder cancer cell lines (T24, 5637, and TCCSUP were from American Type Culture Collection (ATCC, Manassas, VA, USA), EJ was obtained from Japanese Cancer Research Resources Bank (JCRB, Tokyo, Japan), and the BIU-87 bladder cancer cell line from China Center for Type Culture Collection (CCTCC, Wuhan, China)), and an immortalized human bladder epithelial cell line (SV-HUC-1 was from ATCC) were preserved in RPMI 1640 (Gibco, Grand Island, NY) or DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) in an atmosphere of 5% CO2 at 37 °C. These cell lines were authenticated by short tandem-repeat (STR) profiling, and within 4 passages from purchase.
A total of 140 human bladder cancer tissue samples and 34 adjacent bladder epithelial tissue samples were obtained from the General Hospital of Southern Theater Command (China) from February 2016 to October 2019. Inclusion criteria were confirmed non-muscle-invasive bladder cancer (NMIBC, n=70) or muscle-invasive bladder cancer (MIBC, n=70), while the exclusion criterion was metastasis before surgery. All patients provided informed written consent, and the study was approved by the Institute Research Ethics Committee, General Hospital of Southern Theater Command, China, and followed the guidance of Declaration of Helsinki.
Plasmid construction and transfection
Human miR-145-5p mimics and its negative controls (NC mimics), miR-145-5p inhibitors and its negative controls (NC inhibitors) were acquired from Vipotion Biotechnology (Guangzhou, China). Besides, OCT4-pg5 (NR_131184.1), OCT4B (NM_203289.6, ENSG00000236375), OCT4-pg5 siRNAs (si-OCT4-pg5), OCT4B siRNAs (si-OCT4B), miR-145-5p mimics (miR-145), OCT4-pg5 plus miR-145-5p mimics (OCT4-pg5+miR-145-5p), and empty pcDNA3.1(+) (control) were all from Vipotion Biotechnology. Moreover, these PCR products were digested with BamHI//NotI and cloned into the vectors, followed by DNA sequence verification. T24 or 5637 cells were separately transfected with these plasmids using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) for 48h according to the manufacturer’s protocol.
RNA extraction and real-time PCR analysis
Total RNA was extracted from tissues or cells using TRIZOL reagent (Invitrogen Life Technologies) according to the manufacturer's instructions. Real-time PCR was performed using a Stratagene Mx3000P Real-time PCR System (Applied Biosystems, Agilent Stratagene, America) and Bestar qPCR RT Kit (DBI Bioscience, Shanghai, China). The amplification procedure was as follows: 94 °C for 2 min, followed by 40 cycles of 94 °C for 20 s, 58 °C for 20 s, and 72 °C for 20 s. A dissociation step was performed to generate a melting curve for confirmation of amplification specificity. For miR-145-5p, U6 was used as the internal reference, while GAPDH was used as the reference for others. Relative gene expression levels were calculated using the 2-ΔΔCt method. All primers used were designed and produced by Vipotion Biotechnology, and are listed in Table S6.
Luciferase reporter assay
The wild-type 3’UTR sequence of OCT4B containing the putative miR-145-5p binding site was cloned into psiCHECK2 (Promega, Madison, WI, USA) to construct a 3’UTR luciferase reporter. For miRNA target analysis, cell lines (T24 and 5637) were transfected with the luciferase reporter and co-transfected with empty vector (control), miR-145-5p, wild-type OCT4-pg5 (wt-OCT4-pg5), wild-type OCT4-pg5 plus miR-145-5p or empty (control), miR-145-5p, mut-type OCT4-pg5 (mut-OCT4-pg5), or mutant-type OCT4-pg5 plus miR-145-5p plasmid as indicated. The luciferase activities were measured 48 h after transfection using a Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instructions.
After transfection, T24 or 5637 cells were grown on glass chamber slides to 90% confluence, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in phosphate‑buffered saline (PBS) for 30 min, and blocked with 3% bovine serum albumin (BSA) in PBS for 1h at room temperature. Cells were then incubated with anti-OCT4B, anti-β-catenin, anti-E-cadherin, and anti-vimentin antibodies overnight at 4 °C. Immunolabeled cells were incubated with FITC-conjugated secondary antibody (Bioworld, Atlanta, GA, USA) for 1 h and finally counterstained with DAPI for 15 min. Staining patterns were examined and captured using a laser scanning confocal microscope.
Total cellular protein was extracted in lysis buffer (Beyotime; Shanghai, China) and quantified using the Bradford method. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically onto polyvinylidene difluoride membranes (Millipore, USA). The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-OCT4B, anti-N-cadherin, anti-β-catenin (all from Santa Cruz Biotechnology; Dallas, TX, USA), anti-vimentin, anti-E-cadherin, and anti-GAPDH (all from Affbiotech; Shanghai, China). Protein levels were quantified by densitometry using Image-Pro Plus 6.0.
Cell viability assay
Cell proliferation was measured using the Cell Counting Kit-8 (Dojindo, Cat. No.CK04) according to the manufacturer’s instructions. T24 or 5637 cells were seeded in 96-well culture plates at 1×104 cells/well, cultured overnight, transfected with the indicated plasmids for 48 h, washed, and cultivated in complete medium for the indicated growth period (0, 24, 48, and 72 h). Cells were then treated with 10 ml /well Cell Counting Kit-8 solution for 4 h, and total viable cell number estimated by the absorbance at 450 nm using a microplate reader (Thermo Fisher Scientific, Multiskan MK3).
Migration and invasion assays
In vitro migration and invasion assays were conducted using uncoated and Matrigel-coated 24-well transwell chambers (pore size of 8 µM; Costar, Corning, NY, USA), respectively, according to the manufacturer’s instructions. T24 or 5637 cells were plated at 5×105/well in the upper chambers of 24-well transwell plates with FBS-free medium, while the bottom chambers were filled with culture medium containing 20% FBS. After 48 h of incubation at 37 °C in a humidified 5% CO2 atmosphere, cells in the upper chambers were removed, and migratory or invasive cells were stained with 0.1% crystal violet solution for 15 min. Total cell numbers from 6 randomly chosen fields per membrane were quantified at 200× magnification. Mean cell numbers from triplicate assays were calculated for each condition.
After transfection, T24 cells were seeded into 6-well plates at 2×105 cells/well and allowed to grow to 90% confluence in complete medium. Cell monolayers were then wounded using a sterile plastic pipette tip, washed three times with PBS to remove cell debris, and incubated in serum-free medium for 24 h. Cells migrating into the wound area were photographed under an inverted microscopy at designated times, and the average distance of migration was calculated.
Colony formation assays
Transfected T24 or 5637 cells were seeded in 6-well plates at 500 cells/well and cultured for 10 days. Colonies were fixed in paraformaldehyde, stained with crystal violet, photographed, and counted.
Cell cycle and apoptosis analyses
Cell cycle and apoptosis analyses were conducted by flow cytometry (FCM) using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). For cell cycle analysis, transfected cells were plated at 5×105/mL in 6-well plates, incubated for 4–6 h in complete medium, washed with PBS, and then incubated in fresh complete medium for another 48 h. Cells were harvested, centrifuged, and fixed in 70% cold ethanol for 2 h. DNA staining was conducted using 300 ml /well cell cycle staining kit solution (Vazyme Biotech, Nanjing, China) for at least 15 min under darkness. For cell apoptosis analysis, cells transfected as indicated were stained using an AnnexinV-FITC Apoptosis Detection Kit (Vazyme Biotech). The proportion of apoptotic cells was analyzed by FCM using Cell Quest software.
Xenograft tumor model
Six-week-old BALB/c nude mice were acquired from the Model Animal Research Center of Southern Medical University. All animal experiments approved by the Institutional Animal Care and Use Committee of the General Hospital of Southern Theater Command. To establish the xenograft tumor model, 5×106 T24 cells stably transfected with si-OCT4-pg5 or NC-si-RNA, and 5×106 5637 cells stably transfected with pcDNA3.1(+)/OCT4-pg5 or NC-pcDNA3.1(+) were injected subcutaneously in the left flank of separate BALB/c nude mice groups. Tumor volumes were evaluated every three days and calculated according to the equation
where A is the largest diameter and B is the perpendicular diameter. After 27 days, mice were sacrificed and tumors were isolated and weighted.
All statistical analyses were performed using SPSS 22.0, and graphs were constructed using GraphPad Prism 5. Results are expressed as mean ± SD of at least three independent experiments. Group means were compared using independent samples t-test. Categorial data were analyzed by the chi-square test or Fisher exact test. P < 0.05 (two-tailed) was regarded as statistically significant for all tests.