We obtained the RNA sequencing profiles and clinical data of bladder cancer from The Cancer Genome Atlas (TCGA) data portal (https://portal.gdc.cancer.gov/) and the cBio Cancer Genomics Portal (http://cbioportal.org) in August 2019. Another independent dataset GSE13507 from GEO database was also retrieved to validate the differential expressions of C1QTNF6 in bladder cancer and normal tissues, as well as characteristics and prognosis.
The human bladder cancer cell lines (T24, 5637, UMUC3, RT4, and BIU87) and normal urothelial bladder cell line (SV-HUCL) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). T24, BIU87 and 5637 cells were grown in RPMI 1640 medium (Corning Incorporated, Corning, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, Thermo Scientific, Waltham, MA, USA). UM-UC-3 cells were grown in Dulbecco's modified Eagle's medium (DMEM, Corning Incorporated, Corning, NY, USA) containing 10% FBS. RT4 cells were grown in McCoy’s 5A (modified) medium (Boster Biological Technology, Wuhan, China) containing 10% FBS, SV-HUCL cells were grown in F-12K medium (Thermo Scientific, Waltham, MA, USA) containing 10% FBS. All cells were maintained in medium supplemented with 1% penicillin/streptomycin and were then cultured at 37℃ with 5% CO2.
The small interfering RNAs (siRNAs) against C1QTNF6 (siC1QTNF6; siRNA1, GCAACGACUUCGACACCUATT UAGGUGUCGAAGUCGUUGCTT; siRNA2, CCUGAUGUGUGAGAUCCCUTT AGGGAUCUCACACAUCAGGTT) were synthesized by Genepharma (Shanghai, China). Lipofectamine 3000 (Invitrogen, Thermo Scientific, Waltham, MA, USA) was utilized for cell transfection under the manufacturer's instructions.
Cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China ) containing 1% protease inhibitor (Sigma Aldrich, Saint Louis, MO, USA). The protein concentrations were determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China). Total proteins (30-40ug) were separated by 10% SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany ), followed by blocking with 5༅ non-fat milk in Tris-buffer saline with 0.1༅ Tween 20 (TBST) for 1–2 h at room temperature. Then, membranes were incubated overnight at 4 ℃with primary antibody against C1QTNF6 (ab36900, 1:1000 dilution, Abcam, USA); GAPDH (AB0037, 1:5000 dilution, Abways, China). After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (#7074, Cell Signaling Technology, USA). The protein signals were visualized by enhanced chemiluminescence (ECL) chromogenic substrate (Bio-Rad Laboratories, USA). GAPDH was used as an internal control.
Quantitative reverse transcription PCR
Total RNA was extracted using a Trizol reagent (Takara Biotechnology Co., Ltd., China), and cDNA reverse transcription, and qRT-PCR was conducted under the guidance of Reverse Transcription PrimeScript 1st Stand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., China) as well as quantitative PCR reagents SYBR PremixExTaqTM (Takara Biotechnology Co., Ltd., China). β-action was applied as the corresponding internal control. All mRNA levels were evaluated by the 2-ΔΔCT method. Each reaction was performed in triplicate.
In vitro migration and invasion assays
For the migration and invasion analysis, cells were seeded in serum-free medium in the upper chambers of Transwells (Corning Incorporated, Corning, NY, USA). For the migration assay, cells were harvested at 48 h post-transfection and then resuspended in serum-free RPMI 1640; cells (5 × 104/100 µL) were loaded into the upper chamber. The lower chambers contained 600 µL of medium with 10% FBS. The incubation time was 12 h for the T24 cell line and 24 h for the 5637 cell line. After washing with PBS, the cells on the upper surface of the chamber were removed with a cotton swab. The cells on the lower surface of the membrane were fixed with 4%paraformaldehyde for 15 min and stained with a 0.1% crystal violet solution for 15 min, five Transwells fields were photographed with an inverted optical microscope. For the invasion assay, it was performed almost identically to the migration assay except that a Transwells chamber with Matrigel was used instead.
Rabbit polyclonal antibody against C1QTNF6 was obtained from Sigma (College Park, MD, USA). Tissue microarray (TMA) consisting of 54 bladder cancer cases was obtained from OUTDO BIOTECH (Shanghai, China). After deparaffination, rehydration, and antigen retrieval, the TMA slide was incubated with primary rabbit polyclonal anti-human CTRP6 (dilution 1:200; Sigma Antibody; SAB487P) overnight at 4℃. The slide was then incubated with anti-rabbit secondary antibody (ready-to-use solution; Cell Signaling Technology; # 8114), followed by chromogen diaminobenzidine staining. We quantitatively scored the TMA slide using a microscope based on the staining intensity and proportion. Staining intensity of each specimen was scored as follows (negative = 0, weakly positive = 1, moderate positive = 2, and strong positive = 3), and staining percentage as follows (0–10% = 1, 11%–50% = 2, 50–75% = 3, and 75–100% = 4). According to the immunohistochemical scores of C1QTNF6 protein expression, specimens were divided into two groups of high expression group with score great than or equal to 4 and low expression group with score less than 4.
Human Protein Atlas
The Human Pathology Atlas (HPA) project (https://www.proteinatlas.org) included immunohistochemistry data using a tissue microarray-based analysis of 44 different normal tissue types, and proteome analysis of 17 major cancer types[7, 8]. Immunostaining intensity and patient information with the corresponding cancer types were available online. In this study, representative images of protein expression using immunohistochemistry for C1QTNF6 were captured and compared in bladder cancer tissues and normal bladder tissues in the HPA.
Gene set enrichment analysis
Gene set enrichment analysis (GSEA) was applied to evaluate the correlations between C1QTNF6 expression and relevant pathways, using the bladder cancer dataset from TCGA. The detailed protocol for GSEA is available on the Broad Institute Gene Set Enrichment Analysis website (http://www.broad.mit.edu/gsea). Datasets and phenotype label files were created and loaded onto GSEA software (v 4.0.1; Broad Institute, Cambridge, USA). The samples were separated into a high and a low group based on median C1QTNF6 level. The analysis was randomly repeated 1,000 times. A meaningful gene set was defined as an adjusted p-value < 0.05 and a false discovery rate (FDR) < 0.25. Statistical analysis and graphical plotting were conducted using R software (Version 3.3.2).
The statistical differences between groups were analyzed by Student’s t-test or one-way analysis of variance (ANOVA) using GraphPad Prism (GraphPad Software, CA, USA). Survival curves were determined by Kaplan-Meier method. P value < 0.05 was considered significant.