Materials
U-87MG and SHG44 human glioma cell lines were purchased from the Cell Resource Center of the Chinese Academy of Sciences (Shanghai, China).
Cell culture
U87 cells were cultured in DMEM (Hyclone Laboratories, Inc., Beijing, China) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Inc.) in an atmosphere of 5% CO2 at 37°C. The SHG44 cells were cultured in RPMI 1640 (Hyclone Laboratories, Inc.) supplemented with 10% FBS in a 5% CO2 atmosphere at 37°C.
Scratch wound healing assay
The U87MG and SHG44 cells were seeded at a density of 1×105 cells/well in 24-well plates (Corning Life Sciences, Lowell, MA, USA). After 24 h, the cell monolayer was scraped in a straight line using a 20-µl pipette tip and the cells were was hed three times with phosphate-buffered saline. ATRA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored in light-protected vials at −20°C as stock solution. The stock solution was diluted to the desired concentrations immediately prior to use and all experiments were performed under low-light conditions to minimize ATRA photoisomerization. The cells were incubated in low-serum media containing 0.5% FBS and various concentrations of ATRA (5, 10, 20 and 40 µmol/l) and the control group was treated with an equal volume of solvent (DMSO) in culture medium. All the plates were placed in a culture incubator at 37°C and at 0, 6, 12 and 24 h after incubation, the plates were removed from the incubator for images to be captured under a phase-contrast microscope (CKX41; Olympus Corporation, Tokyo, Japan). Image-Pro Plus software (Media Cybernetics, Inc., Rockville, MD, USA) was used to measure the relative migration distances.
Matrigel invasion assay
The U87MG and SHG44 cells were plated at a density of 5×105 cells/well on Transwell chambers (Corning Life Sciences) precoated with 50 µl Matrigel (BD Biosciences, Bedford, MA, USA) diluted with culture medium. Serum-free culture medium containing 5, 10, 20 or 40 µmol/l ATRA was used to incubate the cells of each group and medium containing 20% FBS in the lower chamber served as the chemoattractant. Non-invading cells were removed with cotton swabs after 24 h, while invading cells were dyed with 0.1% crystal violet (Amresco, Inc., Solon, OH, USA) and counted using Image-Pro Plus software under a phase-contrast microscope (magnification, x200). The average cell number in five random visual fields was considered to be the number of invading cells of each chamber.
Flow cytometry
The U87MG and SHG44 cells were seeded at a density of 3×105 cells/well in six-well plates (Corning Life Sciences) with 5, 10, 20 or 40 µmol/l ATRA for 24 h. An Annexin V-fluorescein isothiocyanate cell apoptosis detection kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) was used to detect cell apoptosis and a cell cycle analysis kit (Beyotime Institute of Biotechnology) was used to detect the cell cycle position of each group, according to the manufacturers' instructions. Furthermore, the proliferation index (PI) was used to indicate the proliferation level of each group, according to the following equation: PI = (S + G2/M) / (G0/G1 + S + G2/M) × 100%.
Quantitative polymerase chain reaction (qPCR)
The U87MG and SHG44 cells were seeded at a density of 3×105 cells/well in six-well plates with various concentrations of ATRA for 24 h, as aforementioned. The cells were then lysed and the total RNA was isolated using RNA Fast 200 kit (Shanghai Fastagen Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer's instructions. RNA was reverse-transcribed using a PrimeScript RT Master Mix kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR was performed using SYBR Premix Ex Taq II (Takara Biotechnology Co., Ltd.) on an iQ5 thermal cycler and analyzed using iQ5 software, version 2.0 (Bio-Rad, Hercules, CA, USA). Gene expression was compared using the cycle threshold (Ct). Ct was defined using the following equation: ∆Ct = CtTarget – Ctβ-actin, where β-actin expression was used as the endogenous reference gene. Change in gene expression was evaluated using the 2−∆∆Ct method (14). All primers were designed and synthesized by Takara Biotechnology Co., Ltd. (Table I).
Western blotting
The U87MG and SHG44 cells were cultured in 25 ml cell culture flasks (Corning Life Sciences) with various concentration of ATRA for 24 h. The cells were then harvested for use in assays. The cells were was hed twice with PBS and then scraped in 300 µl radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) with 1 mmol/l phenylmethanesulfonyl fluoride on ice. The lysates were cleared of insoluble material by centrifugation, the protein concentration was determined using a Bradford protein assay kit (Beyotime Institute of Biotechnology) and samples were boiled in 1X SDS-PAGE sample loading buffer, resolved by SDS-PAGE, and then transferred to nitrocellulose membranes (Bio-Rad Laboratories). The membranes were probed with rabbit anti-human MMP-2 (dilution, 1:500; cat. no. BS1236), rabbit anti-human MMP-9 (dilution, 1:500; cat. no. BS1241) and rabbit anti-human GAPDH (dilution, 1:5000; cat. no. ap0063) antibodies, all purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA). Subsequent to was hing in Tris-buffered saline containing 0.02% Tween 20 (Sigma-Aldrich), the membranes were incubated with a secondary polyclonal anti-rabbit IgG antibody conjugated to horseradish peroxidase (dilution, 1:2000; Thermo Fisher Scientific, Pittsburgh, PA, USA). Membranes were developed in Supersignal West Pico chemiluminescent reagent (Pierce Biotechnology, Inc., Rockford, IL, USA).
Statistical analysis
The data are presented as the mean ± standard deviation and were analyzed using SPSS software, version 17.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance was used to compare groups and Fisher's least significant difference tests were performed for subsequent comparisons between groups. P<0.05 was considered to indicate a statistically significant difference.