Patients and specimens
Thirty-two patients who suffered from primary bladder cancer were recruited at Shengjing Hospital of China Medical University from 2013 to 2018. Patients who received pre- or postoperative chemotherapies or targeted therapies were excluded. The study was approved by the Medical Ethics Committee of Shengjing Hospital of China Medical University. All participants provided written informed consent for research purposes and publications. Patients with an average age of 55.63 years (range from 42 to 81 years). Two independent pathologists reviewed all patients' slides to validate the diagnosis and to classify the tumor according to TNM Stage. 13 patients were diagnosed as T1, 9 patients were diagnosed as T2, 8 patients were diagnosed as T3, and 2 patients were diagnosed as T4. Adjacent tissues and tumor tissues were collected simultaneously. All specimens were placed immediately into liquid nitrogen and stored at - 80°C.
LncRNA and miRNA microarray analysis
Total RNA from bladder cancer and adjacent tissues were analyzed with RT2 lncRNA PCR Arrays and miRNA qPCR Assay (QIAGEN NV Corporate, Venlo, The Netherlands). Microarray analysis was performed with the Data Analysis Center as previously described. Data obtained from three independent experiments and RNAs (fold change >1.5 and P-values <0.05) were considered expressed differentially between two groups. All data has been curated by the Gene Expression Omnibus database and the accession number is GSE140584，GSE140585 and GSE140587.
Discovery of lncRNA-miRNA-mRNA associations
Interactions between lncRNA and miRNA were predicted with miRcode, and DIANA-lncBase v.2 followed the previously described[14, 15]. Connections between miRNA and mRNA were predicted with DIANA-TarBase v.8 and miRDB.
Cell culture, transfection reagents, and antibodies
Human urinary bladder cell lines 5637 (HTB-9TM), RT4 (HTB-4TM) and J82 (HTB-1TM) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37◦C and 5% CO2. Control vector pDonor-SH01 (SH025), HAND2-AS1 (DC-W1888-SH01), RARB (A0018), Scrambled control (CSHCTR001-CH1), sh-RARB (HSH070504-CH1) were purchased from GeneCopoeia, Inc. (Rockville, MD, USA). si-negative control, si-HAND2-AS1, Scrambled-miR Control, miR-146 mimic, and miR-146 inhibitor were synthesized by GenePharma Co., Ltd. (Shanghai, China). Antibodies against RARB (sc-514585), COX-2 (sc-376861), Caspase 3 (sc-7272) and GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology, Inc (Dallas, Texas, USA).
Cell proliferation analysis
5x103 5637 or RT4 cells/well were allowed to attach 96-well plates overnight and 20μl of a 5mg/ml stock of methylthiazolyldiphenyl-tetrazolium bromide (MTT, 298-93-1, Sigma-Aldrich Inc., St. Louis, MO, USA) was added to the medium post-treatment for 0, 12, 24, 36, or 48 h. After 4 h, the supernatant was discarded and the pellet was dissolved in 150μl of Dimethyl Sulfoxide (CAS 67-68-5, Santa Cruz Biotechnology, Inc. Dallas, Texas, USA). A microplate reader (Bio-Rad 680 XR, Hercules, CA, USA) was used to detect the absorbance at 490 nm.
1.5x105 cells per well were seeded in 6-well plates. After 12 h, experimental treatments were performed. Cells were stained with the ANNEXIN V-PI (sc-4252 AK, Santa Cruz Biotechnology, Inc. Dallas, Texas, USA) followed the manufacture’s instruction. The fluorescence in thrice independent experiments were determined by flow cytometry.
Tissues and cells were washed twice with pre-cooled PBS and lysed with lysis buffer for 30 min on ice. The concentrations of protein content were determined using a BCA protein assay kit (23225, Pierce; Thermo Fisher Scientific Inc., Waltham, MA, USA). Equal amounts of 25µg proteins were resolved by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in TBS-T for one h at room temperature and incubated with specific primary antibodies (1:1000) at 4 °C with gentle shaking overnight. Membranes were washed three times with TBS-T following incubation with secondary antibodies conjugated to HRP (1:2500; CatLog 31430 and CatLog 31460, Thermo Fisher Scientific Inc., Waltham, MA, USA). The complexes of antibody: protein was detected by enhanced chemiluminescence (Pierce; Thermo Fisher Scientific Inc., Waltham, MA, USA).
Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted by 1ml TRIzol (Merck & Co., Inc., Kenilworth, NJ, USA) according to the manufacturer’s protocol. 1 mg RNA was reverse transcribed to cDNA in 20μl system by First Strand cDNA Synthesis Kit for RT-PCR (Merck & Co., Inc., Kenilworth, NJ, USA). Real-time PCR was performed using the Mx3000P real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). PCR was carried out as follows: 40 cycles of 94 °C for 15s, 60 °C for 10s, and 72 °C for 20s. All procedures were performed independently three times. Gene expression was normalized to the GAPDH to calculate relative expression using the 2-ΔΔCq method. The primer sequences used in this study were listed as below: HAND2-AS1, forward: 5’-GGGTGTTTACGTAGACCAGAACC-3’; reverse: 5’-CTTCCAAAAGCCTTCTGCCTTAG-3’; GAPDH: forward:5’-GTCTCCTCTGACTTCAACAGCG-3’; reverse: 5’-ACCACCCTGTTGCTGTAGCCAA-3’; miR-146: forward: 5’-GAGAACTGAATTCCATGG-3’; reverse: 5’- GAACATGTCTGCGTATCTC-3’; U6: forward: 5’- CGAGCACAGAATCGCTTCA-3’; reverse: 5’-CTCGCTTCGGCAGCACATAT-3’; RARB, forward: 5’- GGTTTCACTGGCTTGACCATCG-3’; reverse: 5’-CCGTCTGAGAAAGTCATGGTGTC-3’; COX-2, forward: 5’- CGGTGAAACTCTGGCTAGACAG-3’; reverse: 5’- GCAAACCGTAGATGCTCAGGGA-3’; Caspase 3, forward: 5’-TGGCCCTGAAATACGAAGTC-3’; reverse: 5’- GGCAGTAGTCGACTCTGAAG-3’.
Dual-luciferase reporter assay
Dual-luciferase activity assay was performed as described previously. The RARB or RARB Mut 3’-UTR was amplified and inserted into the pMIR-REPORTTM vector (Thermo Fisher Scientific, Waltham, MA, USA) followed the manufacturer’s direction. 1×104 cells were plated in a 96-well plate and were allowed to attach overnight. miR-146 mimic or scrambled-miR control was transfected into cells combined with 100 ng of RARB or RARB Mut 3’-UTR. Luciferase activity was determined with the dual-luciferase reporter assay system post-24 h transfection with the Luciferase Reporter Assay System (Promega, Madison, WI, USA).
Generation of 5637 human bladder cancer mouse models
All studies involved experimental animals were approved by the medical ethics committee of Shengjing Hospital of China Medical University and conducted according to the guidelines of the center of experimental animals of Shengjing Hospital of China Medical University. Female BABL/c athymic nude mice (five-week-old) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and housed under specific-pathogen-free conditions. Cells expressing HAND2-AS1, miR-146, or vector stably were subcutaneously injected into the right flanks of mice. Eighteen mice harbored approximately 100mm3 tumors were divided into three groups randomly (six per group) from day 0. The tumors volumes were measured and recorded every four days. Twenty-eight days later, all mice were euthanized, and tumors were resected, fixed and weighed.
The data from all experiments were presented as means plus standard deviation. The association between lncRNA HAND2-AS1 and miR-146 expression was analyzed by Spearman’s correlation coefficient. The differences were evaluated by One Way Analysis of Variance (ANOVA) with LSD test or by Two-way ANOVA with Tukey's multiple comparisons test, and P < 0.05 indicated by ‘*’ or ‘#’ respectively were statistically significant. Statistical analysis was conducted using GraphPad version 7.0 (San Diego, CA, USA).