Compliance with ethical standards
The experiments involving human beings were implemented with ratification of Ethics Committee of Suqian First Hospital by conforming to the principles outlined in the Declaration of Helsinki. Ethical agreements were obtained from the donors or their families through written informed consent. Animal experiments were ratified by Animal Ethics Committee of Suqian First Hospital and concurred with the Guidelines for Animal Experiments of Peking University Health Science Center.
Study subject
Forty patients with bladder cancer undergoing radical cystectomy in Suqian First Hospital from June 2016 to June 2017 were enrolled. Fresh bladder cancer tissues and corresponding adjacent normal tissues were preserved in liquid nitrogen immediately subsequent to resection. None of patients received preoperative radiotherapy, chemotherapy or immunotherapy. Follow-up information was obtained from outpatient clinics and regular telephone interviews.
Bioinformatics methods
Gene Expression Profiling Interactive Analysis (GEPIA) was adopted to analyze the BLCA dataset of The Cancer Genome Atlas (TCGA) database to obtain the genes with significant differences (p < 0.05), from which the genes with |logFC| > 0.5 were screened out. The Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/gds) was also analyzed by using "limma" package (http://www.bioconductor.org/packages/release/bioc/html/limma.html) of the R language with |logFC| > 0.5 and p < 0.05 as thresholds for differential analysis of bladder cancer microarray data GSE65635 and GSE40355. There were 12 samples in microarray data GSE65635, including 4 normal samples and 8 bladder cancer samples. There were 24 samples in microarray data GSE40355, including 8 normal samples and 16 bladder cancer samples. Human lncRNA names were obtained from GENCODE, followed by obtaining of the intersection of significantly differential genes and lncRNA names. Venn diagram was drawn to screen out the lncRNAs among intersection. LncRNA expression trends were collected in Ualcan, and the key lncRNA was determined by comparing the expression trends and combining with the existing literature. The possible downstream miR of the key lncRNA was discovered by starBase and their binding sites were obtained. The databases TargetScan (Cumulative weighted context++ score < 0), DIANA TOOLS (miTG score > 0.6), microRNA (conservation > 0.65, energy < -14, Mirsvr_score < -0.65), and mirDIP (Integrated Score > 0.1) was applied to predict downstream genes of miR. Intersection of downstream genes with significantly differential genes was taken to obtain critical downstream gene. The relevant genes of the critical downstream gene were predicted in GeneMANIA (http://genemania.org/), followed by construction of protein-protein interaction (PPI) network. The most core genes in PPI network were chosen as the key gene, and the binding sites of the miR to the gene were predicted by TargetScan.
Fluorescence in situ hybridization (FISH)
SNHG1 cDNA fragments were amplified from the SNHG1 plasmid as templates by utilizing high fidelity DNA polymerase (Takara, Kyoto, Japan). Based on this template, fluorescein-labeled lncTCF7 FISH probe DNA was prepared with fluorescein-12-dUTP (Roche, Mannheim, Germany) and Klenow DNA polymerase as per the manufacturer's protocol. Four-µm frozen sections were made from bladder cancer tissues and adjacent normal tissues. Subsequent to 5-min immersion in proteinase K, the slides were washed twice in 2 × saline sodium citrate (SSC). The FISH hybridization solution encompassing 30 ng/µL lncTCF7 FISH probe DNA (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) was dripped onto the tissue sections before 16-h incubation at 37℃. The slides were then washed in 0.4 × SSC/0.001% NP-40 for 5 min at 56℃, followed by another 2-min washing in 0.4 × SSC/0.001% NP-40. After being dripped with 4',6-Diamidino-2-Phenylindole (DAPI)-encompassing sealing agent, the slide was mounted and observed under a fluorescence microscope (Olympus, Tokyo, Japan).
Cell incubation
Human normal urothelial cell line SV-HUC-1 (ATCC® CRL-9520), and bladder cancer cell lines [5637 (ATCC® HTB-9), T24 (ATCC® HTB-4™), SW780 (ATCC® CRL-2169™), and UM-UC-3 (ATCC® CRL-1749™) were attained from American Type Culture Collection (ATCC, Manassas, VA, USA). The medium used for SV-HUC-1 was ATCC-formulated F-12K Medium (Catalog No. 30-2004) encompassing 10% fetal bovine serum (FBS, ATCC 30-2020). The medium used for 5637 was ATCC-formulated RPMI-1640 Medium (ATCC 30-2001) encompassing 10% FBS. The medium for T24 was ATCC-formulated McCoy's 5A Medium Modified (Catalog No. 30-2007) with 10% FBS. The medium for SW780 was ATCC-formulated Leibovitz's L-15 Medium (Catalog No. 30-2008) with 10% FBS. The medium for UM-UC-3 was ATCC-formulated Eagle's Minimum Essential Medium (Catalog No. 30-2003) with 10% FBS. All media for cell lines encompassed 100 μg/mL streptomycin and 100 U/mL penicillin. Cell culture was performed at 37℃ with 5% CO2. The media were positioned in humid air and replaced every 2-3 days according to the growth of cells. Cells were subcultured when 80%-90% of the culture plate was covered by cells. Cells were utilized when they reached the logarithmic growth stage.
Cell transfection
Lentiviruses expressing specific targeted knockdown SNHG1 [short hairpin-SNHG1 (sh-SNHG1)] and MDM2 (sh-MDM2) sequences and a scramble shRNA (sh-NC; control shRNA) were constructed by GenePharma (Shanghai, China) (Table 2). Lentiviruses overexpressing SNHG1 (oe-SNHG1), MDM2 (oe-MDM2), and PPARγ (oe-PPARγ), oe-NC, Inhibitor NC, and miR-9-3p inhibitor were obtained from GenePharma. Transfection was implemented using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Subsequent to isolation using RNeasy Mini Kit (Qiagen, Valencia, CA, USA), total RNA underwent reverse transcription to generate cDNA using First Strand cDNA Synthesis Kit (RR047A, Takara). For the detection of miR, the cDNA was obtained by reverse transcription using the miRNA First Strand cDNA Synthesis (Tailing Reaction) kit (B532451-0020, Sangon, Shanghai, China). RT-qPCR reactions were performed using SYBR® Premix Ex TaqTM II (Perfect Real Time) kit (DRR081, Takara) on real-time fluorescence quantitative PCR instrument (ABI 7500, Applied Biosystems, Foster City, CA, USA). The universal reverse primers for miR and the upstream primers for U6 internal reference were provided in the miRNA First Strand cDNA Synthesis (Tailing Reaction) kit, and the other primers were synthesized by Sangon. (Table 3). After recording of the Ct value of each well, the relative expression of mRNAs or miR was calculated using the 2-ΔΔCt method by normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or U6 expression.
Cell counting kit (CCK)-8 assay
The transfected T24 and 5367 cells were resuspended and seeded in 96-well plates at 2 × 103/100 µL/well. Cell viability was evaluated by CCK-8 (Dojindo Laboratories, Kumamoto, Japan) method at 0, 24, 48, 72 and 96 h after seeding. The 10 µL CCK-8 solution was supplemented in each test for 4-h incubation before absorbance measurement at 450 nm with a microplate reader.
5-ethynyl-2’-deoxyuridine (EdU) assay
The cells to be tested were seeded in 24-well plates with three duplicated wells set for cells in each group. EdU (Invitrogen) was supplemented to the medium to achieve a concentration of 10 µmol/L. The medium was discarded subsequent to 2-h culture. Cells received 15-min phosphate buffer saline (PBS) encompassing 4% paraformaldehyde fixing at ambient temperature before 20-min incubation at ambient temperature with PBS encompassing 0.5% Triton-100. Each well was supplemented with 100 µL dye solution before 30-min culture in the dark at ambient temperature. DAPI was added for 5-min nuclear staining. After sealing, 6-10 fields of view were randomly observed under a fluorescence microscope (FM-600, Shanghai Pudan Optical Instrument Co., Ltd., Shanghai, China), and the number of positive cells in each field was recorded.
Flow cytometry
Subsequent to 48-h transfection, the cell concentration was changed to 1 × 106 cells/mL. Subsequent to cell fixing with 70% precooled ethanol solution at 4℃, 100 μL cell suspension (no less than 1 × 106 cells/mL) was resuspended in 200 μL binding buffer. Subsequently, 15-min cells staining were implemented with 10 μL Annexin V-fluoresceinisothiocyanat and 5 μL propidium iodide at ambient temperature under dark conditions. After 300 μL of binding buffer was added, apoptosis was assessed on a flow cytometer at excitation wavelength of 488 nm (2 × 104 cells each time).
Western blot analysis
Subsequent to trypsin treatment, cells were lysed with enhanced radio-immunoprecipitation assay (RIPA) lysis encompassing protease inhibitors (BOSTER, Wuhan, Hubei, China), followed by estimation of protein concentration using Bicinchoninic Acid (BCA) Protein Quantification Kit (BOSTER). Proteins underwent separation by 10% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE). Then, the separated proteins were electroblotted into a polyvinylidene fluoride (PVDF) membrane which was sealed by 5% bovine serum albumin to block nonspecific binding. Overnight cell incubation was conducted at 4℃ after supplementation with primary rabbit antibodies (Abcam, Cambridge, UK) to Cleaved caspase-3 (ab49822, 1: 500), Bcl-2-Associated X (Bax, ab32503, 1: 1000), B-cell lymphoma-2 (Bcl-2, ab196495, 1: 500), MDM2 (ab226939, 1: 3000), PPARγ (ab45036, 1: 500), Ubiquitin (ab7780, 1: 2000), and β-actin (ab8227, 1: 500). Then, horseradish peroxidase-tagged goat anti-rabbit secondary antibodies (ab205719, 1: 2000, Abcam) were supplemented for 1-h membrane incubation at 4℃. After development in ECL working fluid (EMD Millipore Corporation, Billerica, USA), the bands in western blot images were quantified by Image J analysis software by normalizing to β-actin.
RNA pull down
Cells were transfected with biotinylated wild type (WT) miR-9-3p and mutant type (MUT) miR-9-3p (50 nM each). After 48 h of transfection, 10-min cell incubation was implemented with specific cell lysis (Ambion, Austin, Texas, USA). Then, 3-h lysate incubation was conducted with M-280 streptavidin magnetic beads (Sigma, St. Louis, MO, USA) pre-coated with RNase-free and yeast tRNA at 4℃ before two cell washes in cold lysis and RT-qPCR detection of SNHG1 expression.
RNA immunoprecipitation (RIP) assay
The binding of miR-9-3p to MDM2 was detected by RIP kit (Millipore, Temecula, CA, USA). Briefly, 5-min cell lysing was implemented in an ice bath with equal volume of RIPA lysis (P0013B, Beyotime, Shanghai, China), and supernatant was removed subsequent to 10-min centrifugation at 14000 rpm and 4℃. A portion of the cell extract was applied as input, and a portion was co-precipitated with antibody. RNA exaction was implemented by treating samples with proteinase K for subsequent RT-qPCR detection of MDM2. Antibodies used for RIP were as follows: rabbit anti-Argonaute 2 (AGO2) (1: 100, ab32381, Abcam) was mixed at ambient temperature for 30 min, and rabbit anti-human Immunoglobulin G (IgG; 1: 100, ab109489, Abcam) was applied as a NC.
Dual luciferase reporter gene assay
The synthesized MDM2 3’ untranslated region (UTR) gene fragment MDM2-WT and the MDM2-MUT mutated at the binding site were constructed into a pMIR-reporter plasmid (Beijing Huayueyang Biotechnology, Beijing, China). Luciferase reporter plasmids were co-transfected with miR-9-3p into HEK293T cells (Shanghai Beinuo Biotechnology, Shanghai, China). Forty-eight h subsequent to transfection, cells were lysed, and detected using a luciferase detection kit (K801-200; Biovision, Mountain View, CA, USA).
Immunoprecipitation (IP)
Cells were lysed in lysis buffer [mixture of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1 mM ethylene diamine tetraacetic acid, 0.5% NP-40, and protease inhibitor], and cell debris was cleared by centrifugation. After the concentration of lysis was measured by BCA, the same amount of protein was taken from each experimental group and replenished to the same volume with cell lysate. Afterwards, 1 μg anti-MDM2 (ab226939, 1: 100, Abcam), PPARγ (ab45036, 1: 100, Abcam) and 15 μL protein A/G beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were added for 2-h incubation. Subsequent to three washes with cell lysis, beads were collected by centrifugation, added into an equal volume of reductive loading buffer, and boiled at 100℃ for 5 min. Subsequent to SDS-PAGE, samples were electroblotted to PVDF membranes (Millipore), and then analyzed by immunoblotting.
Subcutaneous tumorigenesis model in nude mice
Healthy nude mice aged 6-8 weeks (Beijing Institute of Pharmacology, Chinese Academy of Medical Sciences, Beijing, China) were bred in specific pathogen-free animal laboratory with 60%-65% humidity at 22-25℃. They were fed in separate cages under 12:12-h light-dark cycle with food and water available ad libitum. The experiment was started one week after acclimation, and the health status of nude mice was observed before the experiment. Approximately 2 × 106 cells were suspended in 200 μL PBS, and then subcutaneously injected into the left or right hindlimbs of nude mice (10 mice/group). At 28 days subsequent to injection, mice were euthanized, followed by measurement and weighing of tumors.
Statistical analysis
All measurement data were manifested as mean ± standard deviation and analyzed by SPSS 21.0 software (IBM, Armonk, NY, USA), with p < 0.05 as a level of statistically significance. If data conformed to normal distribution and homogeneity of variance, data within groups were compared by paired t test, while data between two groups were compared by unpaired t test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) or repeated measures ANOVA. Intra-group pairwise comparison was performed using post-hoc test. Rank sum test was performed if data did not conform to normal distribution or homogeneity of variance. Kaplan-Meier was adopted to calculate patient survival curves, and log-rank was utilized to analyze patient survival differences.