Cell culture and TGFβ1 treatment
Human bladder cancer cell lines (RT4 and T24) (Shanghai Cell Bank, China) were propagated in DMEM (Invitrogen) supplemented with 10% FCS at 37°C in 5% CO2 cell culture incubator. In the TGFβ1 (Sigma Aldrich, St. Louis, MO) treatment, the cells were serum starved overnight and treated with 2.5 ng/ml TGFβ1 for 24 hours. The medium containing TGFβ1 was replaced every 24 hours (The Clinical Research Ethics Committee of Central South University approved the research protocols, and written informed consent was obtained from the participants).
microRNA and transient transfection
miR-221 mimics, control mimics, miR-221 inhibitors, and control inhibitors were purchased from RiboBio (Guangzhou, China). RT4 and T24 cells were seeded into 6-well plates until 50%–60% confluent and then transiently transfected with 60 nM control or miR-221 mimics or with 120 nM control or miR-221 inhibitors using the X-treme GENE siRNA Transfection Reagent (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. After 48 hours of miRNA transfection, the cells were harvested for further study.
Quantitative real-time PCR
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. For mRNA detection, first-strand cDNA was synthesized using a PrimeScript RT reagent kit (Perfect Real Time; Takara, Dalian, China). Quantitative real-time PCR was performed using a SYBR Premix Ex Taq™ II kit (Takara, Dalian, China) on a CFX96 real-time PCR system (Bio- Rad, Hercules, CA, USA). The PCR conditions were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 34 s. β-Actin was used as an internal control to normalize the results. For miRNA detection, miR-221 levels were determined using a TaqMan microRNA kit (Applied Biosystems) and normalized to small nuclear RNA (Rnu6), which served as a control; the data were expressed as the log 2 fold change in respective miR/U6 snRNA levels. Primers for miR-221 and U6 reverse transcription and amplification were designed by and purchased from RiboBio Co., Ltd. (Guangzhou, China).
Western blot analysis
Whole cell extracts were prepared with a cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manual, and then, the protein was quantified by a BCA assay (Pierce, Rockford, IL, USA). Then, the protein samples were separated by SDS-PAGE (10%) and detected by Western blot using polyclonal (rabbit) anti-STMN1, anti-Fibroactin, anti-N-Cadherin, anti-E-Cadherin and anti-Vimentin antibody (Santa Cruz Bio-technology, Santa Cruz, CA, USA, 1:1000). Goat anti-rabbit IgG (Pierce, Rockford, IL, USA) secondary antibody conjugated to horseradish peroxidase and ECL detection systems (SuperSignal West Femto, Pierce) were used for detection.
Cell survival assay
The 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to estimate cell viability [22]. Briefly, cells were plated at a density of 1 × 104 cells per well in 96-well plates. After exposure to specific treatment, the cells were incubated with MTT at a final concentration of 0.5 mg/ml for 4 h at 37°C. After the removal of the medium, 150 mM DMSO solutions were added to dissolve the formazan crystals. The absorbance was read at 570 nm using a multi-well scanning spectrophotometer reader. Cells in the control group were considered 100% viable.
Invasion assay
Cells were cultivated to 80% confluence on the 12-well plates. Then, we observed the procedures of cellular growth at 24 h. All the experiments were repeated in triplicate. The transwell invasion chambers were used to evaluate cell invasion. Then cells invasing cells across the membrane were counted under a light microscope.
Adhesion assay
Cells were pretreated with or without different concentrations of excisanin A for 24 h. The cells were suspended in serum-free DMEM medium to form a single-cell suspension and were seeded into 96-well plates precoated with Matrigel™ (BD Biosciences, Franklin Lakes, NJ, USA). The wells were incubated at 37°C for 50 min and washed three times with PBS to remove the non-adherent cells. Cell viability was determined via the MTT assay described above.
Wound healing assay
For the wound healing assay, cells were seeded in 12-well plates and grown to 90% confluence. Monolayers in the center of the wells werescraped with pipette tips and washed with PBS. Subsequently, the cellswere cultured in serum-free DMEM medium in the absence or presenceof different concentrations of excisanin A for 24 h. Cell movement intothe wound area was monitored and photographed at 0 and 24 h usinga light microscope. The migration distance between the leading edge ofthe migrating cells and the edge of the wound was compared as previous work [23].
Luciferase reporter assay
HEK293 cells (1 × 104 cells/well) were plated in a 48-well plate and cotransfected with 50 n M of either miR-221 or microRNA control (miRcontrol), 20 ng of either pGL3-STMN1-3′-UTR-WT or pGL3-STMN1-3′-UTR-Mutation, and 2 ng of pRL-TK (Promega, Madison, WI, USA) using Lipofectamine TM 2000 (Invitrogen). The pRL-TK vector was cotransfected as an internal control to correct the differences in both transfection and harvest efficiencies. HEK293 cells were collected 48 h after transfection and assays were performed by using the dual luciferase reporter assay system (Promega).
Statistical analysis
All experiments were performed at least in triplicate, and each experiment was independently performed at least 3 times. Data are presented as the means ± standard deviation (SD) and were analyzed using SPSS 19.0. Statistical significance was assessed using the two-tailed unpaired Student’s t-test. Differences were considered statistically significant when the P value was <0.05.
