We retrieved the microarray expression of miR-1307-5p data from the GEO (Gene Expression Omnibus, https://www.ncbi.nlm.nih.gov/geo/) datasets and TCGA (The Cancer Genome Atlas, https://tcga-data.nci.nih.gov/tcga) database. GSE121711 datasets contains 10 primary bladder cancer tissues and 8 normal bladder tissues. The TCGA bladder cancer datasets included 429 bladder cancer samples after excluding cases with incomplete clinical information. The miR-1307-5p expression profiling in bladder cancer were analyzed using R statistical environment (R version 3.5.1). Patients were divided into high and low miR-1307-5p expression groups -using the median expression level - as the cut-off. The-Kaplan-Meier analysis was performed to for statistical significance in survival rates and generate the overall survival plots. P<0.05 was set as - statistical significance.
A total of 20 pairs of primary bladder cancer tissues and matched noncancerous tissues were collected from bladder cancer patients, who received resection at The First Affiliated Hospital of Nanchang University in 2021. None of the patients received chemotherapy or radiotherapy prior to the surgery. The tumor pathological type was diagnosed by three independent pathologists, and the matched normal bladder epithelial tissues, which were collected from more than 5cm away from the tumors, were simultaneously validated. The study was approved by the Ethics Committee of The First Affiliated Hospital of Nanchang University. Informed consent was obtained from each patient prior to their inclusion in the study.
Cell culture and transfection
A normal human bladder uroepithelium cell line (SV-HUC-1) and bladder cancer cell lines (5637, T24, UMUC-3 and RT-4) used in this study were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) in August 2019. All cell lines were authenticated by short tandem repeat DNA profiling analysis in 2019. SV-HUC-1, 5637 and T24 cells were cultured in RPMI-1640 medium (Procell, Wuhan, China) supplemented with 10% fetal bovine serum and 1% double antibiotics (penicillin and streptomycin, NCM Biotech, China). UMUC-3 and RT-4 cells were cultured in DMEM (Procell, Wuhan, China) with 10% fetal bovine serum and 1% double antibiotics (penicillin and streptomycin, NCM Biotech, China). The cells were incubated in humidified air with 5% CO2 at 37℃. The miR-1307-5p mimics, miR-1307-5p inhibitor, small interference RNAs of MDM4 and their negative controls were obtained from Shanghai GenePharma Co, Ltd (Shanghai, China). Cell transfection was conducted using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Upon 72 hours of transfection, analyses were carried out as indicated.
RNA extraction and real-time RT-qPCR
Total RNA was extracted from cells or tissue samples using TRIzol reagent (Thermo, USA). cDNA synthesis was carried out using the reverse transcription kit (Genecopoeia, Guangzhou, China). The sequence of specific miR-1307-5p reverse transcription primer was shown as follow: 5’ -GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAGCCGG-3’. We used BeyoFastTM SYBR Green PCR kit (Bio-Rad, Shanghai, China) for real-time qPCR, under the following conditions: 1x (- 95℃,2 min) and 40x (95℃,15s; 60℃,30s). The primers for RT-qPCR analysis of miR-1307-5p were as follows: forward, 5’-TCGACCGGACCTCGA-3’, and reverse, 5’-CAGTGCGTGTCGTGGA-3’. MDM4 forward and reverse primers are: 5’-TGATTGTCGAAGAACCATTTCGG-3’ and 5’-TGCAGGGATCAAAAAGTTTGGAG-3’. - U6 small nuclear RNA or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal controls, respectively.
Western blotting analysis
Cells were lysed with RIPA lysis buffer kit (Beyotime, Shanghai, China), and - protein samples were quantified using the BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China). Total protein lysates (25ug) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. Membranes were blocked with 5% skim milk for 1 hour at room temperature, followed by incubation for overnight at 4℃with the primary antibodies: anti-GAPDH (1:6000, Proteintech, Beijing, China), anti-Ki-67 (1:1000, Proteintech, Beijing, China), anti-MDM4 (1:1500, Proteintech, Beijing, China), anti-PCNA (1:2000, Cell Signaling Technology, USA), anti-caspase9 (1:1000, CST, USA), anti-caspase3 (1:1000, CST, USA), anti-YAP (1:5000, Proteintech, Wuhan, China), and anti-LATS1 (1:2000, Proteintech, Wuhan, China). After washing with TBST buffer for three times, membranes were incubated with secondary antibody (1:10000, CST, USA) for 1 hour at 37℃. ECL was used for development and Biolmaging Systems was used for signals detection and visualization.
Cell viability was measured using the Cell Counting Kit-8 assay kit (Sigma, USA). Cell suspensions were seeded in 96-well plates at a density of 3000 cells per-well with 3 repeats for each test. At 0, 24, 48, and 72 hours after inoculation respectively, 100ml CCK-8 was added to each well and incubated for additional 1 hour at 37℃. The absorbance values were measured at 450nm using a microplate reader.
Cell cycle analysis
Cells were digested with trypsin/EDTA (NCM Biotech, China), and washed twice with cold PBS. Then, the collected cells were fixed in pre-cold 70% ethanol for 24 hours at 4℃and incubated with RNase A for 30 min. After that, the cells were stained with propidium iodide (PI, Beyotime, Shanghai, China) for 10min in darkness. Finally, cell cycle was determined with flow cytometry (BD Biosciences) and the percentage of cells in different phases was analyzed using FlowJo 10 software.
Cell apoptosis assay
Cell apoptosis was determined by Annexin V and PI double staining protocol using the Annexin V-FITC apoptosis detection kit (KeyGen Biotech, China). Cells were harvested with trypsin, washed with PBS, pelleted in a cooled centrifuge, and resuspended in binding buffer. Next, cells were incubated with Annexin V-FITC and propidium iodide (PI) and maintained in darkness for 15 min at room temperature. Flow cytometric analysis was performed with a FACS Calibur instrument (BD Biosciences). The results were analyzed by FlowJo 10 software.
in vivoTumorigenicity assay
Twelve six-week-old male BALB/c nude mice were obtained from Shanghai Experimental Animal Center (Shanghai, China), which weighted about 18-20g each. All mice were housed in a strict pathogen-free conditions. All animal experiments were performed using protocols approved by the Animal Center of The First Affiliated Hospital of Nanchang University. A random number table method was used to divide the mice into two groups, negative control group and miR-1307-5p mimics group (N=6 for each group). Each nude mouse was subcutaneously inoculated with 100µL of cell suspension (1x106 cells) in the right axillary sub cutis. The length and width of the tumor was measured every 4 days using a vernier caliper, and the tumor size was calculated as follows: volume (mm3) = 0.5 ×length (mm) ×width2(mm2), and the growth curve was calculated. At the end point, the mice were euthanized, the tumors were collected and weighed. During the experiment, care was taken to minimize the pain of the mice without affecting the results of the experiment. No deaths occurred during the experiment.
Luciferase reporter assay
The luciferase reporter assay was conducted for miRNA target validation. TargetScan (http://www.targetscan.org/vert_72/), a biological prediction website, predicted that 3’-UTR sequences of MDM4 might be the direct target of miR-1307-5p. In order to construct the luciferase plasmid, the MDM4 3’-UTR gene sequence or a mutant sequence were inserted into pGL3 promoter vector (Invitrogen, USA), which was defined as wt-MDM4-3’-UTR and mut-MDM4-3’-UTR. According to the manufacturer's protocol, T24 cells that transfected with miR-1307-5p mimics or miR-NC (negative control) were co-transfected with luciferase reporter plasmid and Renilla luciferase (internal control) vector using Lipofectamine 2000 (Invitrogen, USA). While 5637 cells that transfected with miR-1307-5p inhibitor or miR-NC were co-transfected with luciferase reporter plasmid and Renilla luciferase control vector using Lipofectamine 2000 (Invitrogen, USA). Cells were collected and lysed after 48 hours transfection, followed by analyses using the Dual Luciferase Reporter Assay Kit (Promega, USA); reporter activity was calculated based on firefly luciferase - normalized to Renilla luciferase.
Data were expressed as the mean ± standard deviation (SD). Differences between two independent groups were analyzed using a two-tailed Students’ t-test. Differences between multiple groups were analyzed using ANOVA tests. Survival curves were analyzed using the Kaplan-Meier method and compared using the log-rank test. A p-value < 0.05 was considered statistically significant.