2.1 Patients Tissue Specimens
For this study we obtained 72 pairs of BCa tissues and matched adjacent nontumor bladder tissues from the Second Hospital of Tianjin Medical University between September 1, 2015 and July 31, 2020. All patients received surgical resection without preoperative radiation or chemotherapy, and were pathologically diagnosed as having BCa. Basic demographics and clinicopathological features were also collected. The Ethics Committee of the Second Hospital of Tianjin Medical University approved this study., and all patients signed the informed consent documents.
2.2 Cell Culture
The BCa cell lines 5637, T24, and EJ were purchased at the Chinese Academy of Sciences Cell bank, and the 253J-BV line was a kind donation from Professor Lei Li of the First Affiliated Hospital of Xian Jiaotong University. The cells were cultured as previously described.(Shen et al. 2020)
2.3 Cell Transfection
To construct the circSTK39 plasmids used in this study, we cloned the full length of circSTK39 and inserted it into HBLV plasmids (Hanbio, Shanghai, China). Cell lines were transfected by using lentivirus, and stable cell lines were selected and placed in the medium with 0.5 µg/ml puromycin for 7 days. GenePharma (Shanghai, China) designed small interfering RNA (siRNA), miRNA mimics, miRNA inhibitors, and their matching negative controls. Following the manufacturer's instructions, we used Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA) to transfect siRNAs and miRNA mimics or inhibitors. All sequences included in the investigation are listed in Table S1.
2.4 Cell Proliferation Assay
To explore changes in proliferation ability, cell counting kit-8 (CCK-8) assay and colony formation assay were applied. For the CCK-8 assay, the pretreated BCa cells were counted and added into a 96-well plate, with a cell concentration of 1.5×103 cells/well. The CCK-8 reagent (APExBIO, USA) was then used to detect the optical density at 450 nm after 3 h of incubation at 37°C. For the colony formation experiment, approximately 800 transfected cells per well were planted in six-well plates. After 8–14 days, the cells were fixed in PFA at 4°C and stained with crystal violet at 37°C. Finally, the colonies were counted by hand.
2.5 Wound Healing Assay
A 6-well plate was seeded with 5×105 transfected cells that were then grown for 24–48 h until they formed a cell monolayer. Using a 10 µl pipette tip to scratch the cell layer, we then washed each well with PBS and continued culturing with 1640 medium. Photographs of wound healing assay were taken at the same place at 0 and 24 h after scratching.
2.6 Transwell Assay
For the transwell assay 2×104 transfected BCa cells were mixed in 200 µl serum-free 1640 medium and were added to the upper chambers covered with 50 µl of matrigel or left uncovered. The bottom chambers were surrounded with 1640 medium containing 20% FBS. After incubation of 24 h for migration and 48 h for invasion, at 37°C, the cells in the lower chambers were fixed in PFA at 4°C, stained with crystal violet at 37°C, photographed, and analyzed by ImageJ software.
2.7 RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)
Total RNA was obtained from tissues and cell lines by following the instructions on the total RNA Kit (Omega, Norcross, USA). Here, 2 µg of total RNA was reverse transcribed to cDNA with the RevertAid First Strand cDNA SynthesisKit (Thermo Fisher Scientific, Waltham, USA). Subsequently, RNA expression levels were determined by qRT-PCR with SYBR Green Premix Ex Taq (Takara, Nanjing, China) on an ABI 7900HT rapid real-time PCR system (Applied Biosystems, Waltham, USA). For circRNAs and mRNAs, we employed GAPDH as an internal control, and employed U6 as a miRNA internal control. The relative quantitative value of target gene was determined by the 2−ΔΔCT method. The sequences of all primers are listed in Table S1.
2.8 Protein Extraction and Western Blot Analysis
Total protein was extracted from cells with a mixture containing RIPA lysis buffer and 1% protease inhibitors, and the protein concentration was measured using a BCA kit (Solarbio, Beijing, China). Equal quantities of 30 µg protein from each sample were separated by 10% SDS–PAGE and then transferred to PVDF membranes (Millipore, USA). The primary antibodies used were as follows, anti-NR3C2 (1:500, ABclonal, Wuhan, China), E-cadherin (1:1000, Cell Signaling Technology, USA), N-cadherin (1:1000, Cell Signaling Technology, USA), Vimentin (1:1000, Cell Signaling Technology, USA), and anti-GAPDH (1:5000, BOSTER, Wuhan, China).
2.9 RNase R Treatment, Actinomycin D Treatment, and Subcellular Fractionation
For RNase R assay, 10 µg total RNA from T24 and 253J-BV were added with or without 20 U/µl RNase R (Abm, Canada) for 2 h at 37℃, and for Actinomycin D assay, T24 and 253J-BV were added with 2 µg/ml Actinomycin D (Sigma, USA) for 0, 4, 8, 12, and 24 h. We used an NE-PERTM kit (Thermo Fisher Scientific, Waltham, USA) to extract cytoplasmic and nuclear fractions according to the manufacturer's instructions and then used qRT-PCR to detect circSTK39, U6, and 18s expression levels. Additionally, U6 and 18s were enriched in the cytoplasm and the nucleus, respectively. The sequences of U6 and 18s primers are listed in Table S1.
2.10 RNA Fluorescence In Situ Hybridization (FISH)
GenePharma (Shanghai, China) developed the Cy3-labeled circSTK39 probe and the FAM-labeled miR-135a-5p probe used for this study. These probes were used to locate circSTK39 and miR-135a-5p in tissues and BCa cell lines, and the probes’ sequences are shown in Table S1. The specific steps were carried out according to the manufacturer’s protocols for their fluorescence in situ hybridization kit (GenePharma, Shanghai, China). All images were taken by Olympus Confocal Microscope (Olympus, Japan).
2.11 RNA Immunoprecipitation (RIP)
Approximately 1×107 T24 cells were harvested in a 15 cm dish and RIP assay was conducted utilizing a RIP kit (BersinBio, Guangzhou, China) and following the manufacturer's instructions. Subsequently, 5 µg of the magnetic bead and the anti-Ago2 (BOSTER, Wuhan, China) or negative control IgG (BOSTER, Wuhan, China) were co-incubated with the cell lysate at 4°C overnight. Both a total RNA Kit and miRNA kit (Omega, Norcross, America) were used to obtain related RNA, and the expression levels of circSTK39 and miR-135-5p were determined using qRT-PCR.
2.12 Biotin-Coupled miRNA Capture
For biotin-coupled capture Approximately 5×106 BCa cells (T24 & 253J-BV) were transfected with biotin-miR135a-5p or negative control (NC) by lipofectamine 3000 (Thermo Fisher Scientific, Shanghai, China). After incubation for 24 h, the cells were collected into a 1.5 ml centrifuge tube and then lysed by 500 µl lysis buffer. Subsequently, the cell lysates were incubated with 50 µl of washed streptavidin magnetic beads (Invitrogen) at 4°C for 2 h. Next, the beads were washed and added into 500 µl lysis Buffer (Omega, Norcross, USA). Finally, the expression levels of cricSTK39 and NR3C2 were detected by qRT-PCR analysis.
2.13 Biotin-Coupled CircSTK39 Probe Pull-Down Assay
GenePharma (Shanghai, China) designed biotin-circSTK39 and oligo probes to pull down miRNA. The biotin-circSTK39 and oligo probes were incubated for 2 h at room temperature with washed streptavidin magnetic beads (Invitrogen). Subsequently, 50 µl probe-coated beads were incubated with lysates of 5×106 BCa cells at 4°C overnight. After washing, the RNA complexes that had bound to the beads were extracted by 500 µl lysis Buffer (Omega, Norcross, USA), and the expression level of miRNA was analyzed by qRT-PCR assay.
2.14 Dual-Luciferase Reporter Assay
The mutant-type and wild-type circSTK39 reporter plasmids (circSTK39-mut and circSTK39-wt) and NR3C2 mutant-type and wild-type reporter plasmids (NR3C2-mut and NR3C2-wt) were designed by Hanbio (Shanghai, China). The reporter plasmids and miRNA mimics or NC were co-transfected into cells with Lipofectamine 3000, and the activities of both firefly luciferase (LUC) and Renilla luciferase (RLUC) were measured 48 h after transfection using the GloMax® 20/20 Luminometer (Promega, USA) and a Dual-Luciferase Reporter System Kit (Promega, USA). The relative value of luciferase was also calculated for the wild-type and mutant-type groups.
2.15 Xenograft Experiments In Vivo
All animal studies were approved by the Animal Research Ethics Committee of Tianjin Medical University. For in vivo tumor growth studies, 5×106 transfected T24 cells mixed in 1:1 PBS/Matrigel solution were subsequently injected into 4-week-old male BALB/C nude mice. Tumor size was measured weekly according to the formula Volume = (long diameter x short diameter2) ×0.5. The mice were euthanized four weeks after injection, and the tumor tissues were then weighed.
For the metastasis model in vivo, 2×106 transfected T24 cells were injected through the tail vein into 4-weeks old BALB/C nude mice. Using the in vivo FX Pro small animal imaging system, fluorescent pictures of xenografts in nude mice were acquired seven weeks after injection (Brooke company, USA). Both the tumor tissues in THE tumor growth experiment and the lung tumor tissues in the tumor metastasis experiment were fixed in 4% PFA solution and embedded in paraffin.
To perform the immunohistochemistry (IHC) assay, the paraffin-embedded tissue sections were incubated with the primary antibody NR3C2 (1:200, ABclonal, Wuhan, China) and Ki-67 (1:1000, Zsbio, Beijing, China) at 4°C overnight. Next, we used anti-IgG secondary antibody (1:200, BOSTER, Wuhan, China) to incubate the sections at 37°C for 1 h. Finally, each section was evaluated by at least two independent pathologists.
2.17 Statistical Analysis
All results that we analyzed came from at least three independently repeated experiments. We used SPSS (IBM, version 21.0.0) and Graphpad prism 8.0 to analyze and visualize the data, respectively, which we transformed into means ± standard deviations (SD). Comparisons between two groups were evaluated using Student’s t test with two tails, and one-way analysis of variance (ANOVA) was used to examine the differences between the groups. A Kaplan-Meier survival curve was used to describe the OS distribution. Finally, a p-value < 0.05 was set as the threshold for statistical significance.