Animals and the xenograft tumor model
We used pregnant BALB/c nude mice purchased from the experimental animal center of Nantong University (Certificate No: SYXK (SU) 2012-0031). All animal experiments were conducted according to protocols approved by the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number and suffering of animals used in this study.
Lentivirus encoding Wnt5a and a nontargeting sequence NC were transfected into U87 cells to generate stably transfected cells. For assessing tumor growth in vivo, BALB/c nude mice were randomly assigned to three groups of six each. LV-NC U87 cells (1 × 106), LV-siWnt5a U87 cells (1 × 106) and LV-miR-548ac+sh−Wnt5a cells (5 × 106) were separately resuspended in 150 µl PBS and then subcutaneously inoculated into the flanks of BALA/c nude mice. The tumors were monitored regularly at the indicated time points.
U87 and U251 human glioma cells were purchased from the Chinese Academy of Medical Sciences (Beijing, China) and cultured in Dulbecco’s modified Eagle’s medium/high glucose with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) at 37°C in 5% CO2.
An miR-548ac mimic, miR-548ac inhibitor, related negative control (NC) mimics, and an NC inhibitor were transfected into cells (RiboBio Co. Ltd, Guangzhou, China) using Lipofectamine 3000 reagents (Invitrogen, CA, USA). Stably transfected cells were collected after 48 h of culture. Wnt5a small interfering (si)RNA, and an siRNA NC were purchased from GenePharma (Shanghai, China). The target sequence was 5'-GTTTTGGCCACTGACTGA-3'. A plasmid overexpressing Wnt5a (NM_001377271.1) was inserted in a pcDNA3.1 vector (Shanghai, China), and the empty vector was used as its NC. To explore the association between miRNA-548ac and Wnt5a in GBM, U87 and U251 cells were transfected with miR-548ac mimics or the mimic NC and pcDNA3.1-NC or pcDNA3.1-Wnt5a using Lipofectamine 3000 (Invitrogen, CA, USA). Cells were analyzed 48 h after transfection.
Cells were collected in 0.25% trypsin 48 h after transfection. For cell cycle analysis, the cells were fixed with ice cold 75% ethanol for 24 h and resuspended in phosphate buffered saline (PBS). After adding 500 μl/ml Pharmingen PI/RNase Staining Buffer (BD Biosciences, CA, USA) with incubation for another 30 min at room temperature, the labeled cells were assayed by flow cytometry (BD Biosciences, CA, USA). The distribution of cells in the G0/G1, S, and G2/M phases was analyzed with ModFit LT software (Verity Software House, http://www.vsh.com/products/mflt/).
Cell proliferation assays
Cell proliferation was measured with CCK-8 assay kits in accordance with the manufacturer’s instructions (Beyotime Institute of Biotechnology, Jiangsu, China). In brief, GBM cells were seeded in 96-well plates at 3000 cells/well. CCK8 solution (10 μl) was added, followed by incubated for 2 h at 37°C. The absorbance was evaluated at 450 nm with a SpectraMax M5 microplate reader (Molecular Devices, CA, USA).
Colony formation assays
GBM cells in growth phase were seeded at 1000 cells per well in culture medium. The cells were cultured for 2 weeks at 37°C, then fixed in 4% paraformaldehyde for 10 min. Cell colonies were stained with 0.5% crystal violet for 30 min at room temperature and washed in PBS.
Transwell invasion assays
Transwell assays were performed to detect cell invasion. Cells were transfected for 48 h, and then 2 × 105 cells were plated in the upper chamber (with Matrigel for invasion assays) of a 24-well Transwell plate, and 600 μl of FBS was added to the lower chamber. After incubation for 24 h, cells on the upper surface were removed, and cells migrating to the lower chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. For each assay, cells were counted in six random fields.
Wound healing assays
Wound healing assays were performed to analyze cell migration. Briefly, GBM cells were seeded into six-well plates and cultured at 37℃ for 24 h. A linear wound was scratched with a 1 ml pipette tip, and the wells were washed twice with PBS to remove suspended cells and debris. The cells were cultured in medium containing 10% FBS, then the wounds were photographed and the size was measured at 0 h, 24 h and 48 h.
RNA extraction and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis
Total RNA was isolated with TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions and reverse transcribed to cDNA with the SuperScrip III First-strand Synthesis System (Thermo Fisher Scientific, MA, USA). For qRT-PCR analysis, the relative quantification of Wnt5a expression was calculated by the comparative CT method (ΔΔCT). The Wnt5a forward primer was 5′-TCGACTATGGCTACCGCTTT-3′and the reverse primer was 5′-CACTCTCGTAGGAGCCCTT-3′.
The miR-548ac forward primer was 5′-CAAAAACCGGCAATTACTTTTG-3′ and the reverse primer was 5′-CTCAACTGGTGTCGTGGA-3′. The GAPDH forward primer was 5′-ATTCCATGGCACCGTCAAGGCTGA-3′ and the reverse primer was 5′-TTCTCCATGGTGGTGAAGACGCCA-3′.
Luciferase reporter assays
Potential miR-548ac binding sites in the Wnt5a 3´-UTR were predicted with the bioinformatics tools miRwalk (http://starbase.sysu.edu.cn/), miRDB (http://mirdb.org/) and TargetScan (http://www.targetscan.org/). The sites of the predicted target gene and a mutant variant were synthesized and cloned in the pmirGLO Dual-Luciferase miRNA target expression vector (GenePharma, Shanghai, China). GBM cells were seeded in 96-well plates and co-transfected 12 h later with plasmids or miR-548ac mimics with Lipofectamine 3000. The cells were harvested 48 h after transfection, and firefly and Renilla luciferase activity was analyzed with a dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
Western blot analysis
Cells were lysed and proteins were isolated in ice cold RIPA buffer (Thermo, MA, USA). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% skim milk, the membranes were incubated with p-ERK1/2, total ERK1/2, total RAF, p-RAF, Wnt5a, and β-actin primary antibodies purchased from Abcam.
Prism 6 statistical software was used for statistical analysis. All data are presented as the mean ± standard error of mean (SEM) from at least three independent replicates. Statistical analysis of data was performed with Student’s t-test. Differences were considered statistically significant at P < 0.05.