Clinical Specimens
Twenty osteosarcoma tissues and their matched adjacent tissues were collected from patients (15 males and 5 females; average age 19 years old) who received surgical treatment at the Shaanxi Provincial People's Hospital from February 2017 to November 2018. All the patients did not receive radiotherapy and/or chemotherapy before operation and gave informed consent before surgery. The histological diagnosis of osteosarcoma conformed to the World Health Organization's histological criteria for osteosarcoma and the protocols were approved by the Ethics Committee of Shaanxi Provincial People's Hospital. Tissues were frozen in liquid nitrogen immediately and stored at -80℃.
Cell Lines and Culture
Human normal osteoblast cells (hFOB1.19) and human osteosarcoma cell lines (Saos2, 143B, MG-63, U2OS, HOS) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were incubated in DMEM (Gibco, Rockville, MD) containing 10 % fetal bovine serum (Gibco, Rockville, MD) and 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma, St. Louis, MO, USA) at 37 °C in a humidified atmosphere with 5 % CO2.
Quantitative real-time PCR (qPCR)
Total RNA was separated from tissues and cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, tissues or cells were lysed with 600 μL of Trizol, then the supernatant was precipitated with 600 μL of chloroform and isopropanol and washed with 75% ethanol. Finally, RNA precipitation was dissolved with RNase-free H2O. RNA purity and integrity were analyzed by a spectrophotometer and Bio-Rad Experion automatic electrophoresis system (Bio-Rad, CA, USA). The RNA sample was reversely transcribed into cDNA using a PrimeScript RT reagent Kit (Takara Biotechnology, Dalian, China) and cDNA amplification was carried out using TaqManTM MicroRNA Reverse Transcription Kit (Applied Biosystems) and PrimeScript RT Master Mix (Takara, Dalian, China). Then 25-μL reaction system was built, containing 12.5 μL SYBR Premix Ex Taq II, 1.0 μL primers (10 μM) and 1 μL cDNA sample (about 200 ng), and 10.5 μL ddH2O. Each reaction was performed in triplicate wells. PCR program was conducted on a Real-Time PCR System (ABI7500, Applied Biosystems, Waltham, MA, USA) under the following conditions: 95 °C for 1 min, followed by 35 cycles of 95 °C for 20 s, then 56 °C for 10 s and 72 °C for 15 s. All abundances of the transcripts were normalized to U6 or GAPDH. The relative expression of gene was calculated with the 2-∆∆Ct method. All primer sequences were provided in Supplementary Table 1.
Cell Transfection
Human GAS5 gene was amplified by PCR from hFOB1.19 cells and the cDNA sequences were subcloned into pcDNA3.1 vector (Sangon, Shanghai, China) to construct pcDNA-GAS5 plasmid, with the empty pcDNA3.1 vector serving as the negative control (Vector). Plasmids were transfected into U2OS and HOS cells using Lipofectamine 3000 Transfection Reagent, according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). MiR-23a-3p mimic, inhibitor, and its corresponding negative control mimic or inhibitor (NC-mimic and NC-inhibitor) were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In addition, siRNA negative control (Scramble), si-GAS5, and si-PTEN (All obtained from Sangon, Shanghai, China) were transfected into cells as described above.
Analysis of Cell Proliferation
Cell proliferation was detected using CCK-8 assay. Briefly, cells were seeded into 96-well plates and cultured for 24 h. After transfection for 0, 24, 48 and 72 h, 10 μL CCK-8 solution was added into each well and co-incubated with cells for another 2 h at 37 °C. The absorption values were detected at the wavelength of 450 nm with a micro-plate analyzer (Molecular Devices, Sunnyvale, CA, USA).
Invasion Assays
Cell invasion was determined using MatrigelTM Invasion Chambers. Briefly, 1 × 105 cells in 200 µL free-serum DMEM medium were seeded into the upper compartment of 24-well plates, while 600 µL medium, containing 20 % FBS, was added to the lower chamber. After incubation for 48 h, cells on the upper membrane surface were removed using a cotton swab and invaded cells were fixed with 70 % ethanol for 10 min and stained with 0.1 % crystal violet for 15 min. These invasive cells were counted from five randomly selected fields using a light microscope (magnification, 200×, Tokyo).
Cell Apoptosis
Flow cytometry was used to evaluate cell apoptosis. After transfection, cells were collected and washed with phosphate-buffered saline for three times. Cells were stained with 5 μL of Annexin V-FITC and 5 μL of propidium iodide at room temperature in the dark for 20 min, cell apoptosis were examined using flow cytometry (BD Biosciences, USA).
RNA Immunoprecipitation (RIP) and RNA pull-down
RNA immunoprecipitation (RIP) assay was performed to explore the interaction between GAS5 and miR-23a-3p with EZ-Magna RIP RNA-binding protein immunoprecipitation kit (Millipore). After cells were lysed with RIP lysis buffer, 100 μL of the lysate was incubated with RIP immunoprecipitation buffer containing magnetic beads, which were conjugated with human anti-Argonaute2 (Ago2) antibodies (Millipore, Billerica, MA, USA) or control normal mouse immunoglobulin G (IgG; Millipore, Billerica, MA, USA). Among the antibodies, IgG was considered as a negative control. Proteinase K buffer was then added to the samples to digest protein. Finally, the target RNA was extracted and purified for further study by using qPCR.
RNA pull-down assay was performed to examine the interaction between GAS5 and miR-23a-3p. Briefly, cells were transfected with biotinylated miRNA and collected. Then cell lysates were incubated with M-280 streptavidin magnetic beads (Invitrogen, San Diego, CA, USA). The bound RNAs were purified using Trizol reagent (Invitrogen) for further qPCR analysis (the details about the amplified fragment and its primers were shown in Supplementary figure 1B).
Luciferase Reporter Gene Assay
The luciferase reporter gene assay was conducted on the basis of manufacturer’s instructions. A putative miR-23a-3p target binding DNA sequence was amplified by using PCR. We then subcloned into a pmirGLO Reporter plasmid (Promega, Madison, WI, USA) to construct the wild-type GAS5 3′-UTR (GAS5-WT), mutant GAS5 3′-UTR (GAS5-Mut), wild-type PTEN 3′-UTR (PTEN-WT) and mutant PTEN 3′-UTR (PTEN-Mut). Luciferase reporter plasmids and miR-23a-3p mimic or NC-mimic were co-transfected into HEK-293T cells using Lipofectamine 3000. After transfection for 48 h, relative luciferase activity was measured by using a Dual-Luciferase Reporter Assay System (Promega, Madison, Wisconsin, WI, USA).
Western blotting
After transfection, cells were harvested and lysed in Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China) for 30 min at 4 °C. Protein concentration was quantified using an Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were separated on 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5 % skim milk for 1 h at room temperature, the blots were probed with primary antibodies (PTEN, 1:2000 dilution; PI3K, 1:0000 dilution; phosphorylated PI3K, p-PI3K, 1:0000 dilution; AKT, 1:0000 dilution; phosphorylated AKT, p-AKT, 1:2000 dilution; Abcam, Cambridge, UK) overnight at 4°C. Then the membranes were washed and incubated with secondary antibodies (1:2000 dilution; Abcam, Cambridge, MA, USA) for 1 h at room temperature. Then, the chemiluminescent reagent was added to the membrane in an even manner and developed the image with a developing solution. All western blots were subjected to relative optical density (OD) analysis during the experiment. Finally, the signals were visualized using a chemiluminescence imaging system (Bioshine ChemiQ 4800 mini, China, Oxiang, Shanghai).
Tumor Xenograft Assay
All animals were approved by the Ethics Committee for the Use and Care of Animals of Shaanxi Provincial People's Hospital. Female BALB/c nude mice (age:4 weeks old; weight: 16-20 g) were purchased from Institute of Laboratory Animals in Chinses Academy of Medical Sciences (Shanghai, China). The mice were randomly divided into two groups, and U2OS cells (2 × 106) containing with pcDNA-GAS5 plasmid or pcDNA3.1 empty vector were subcutaneously injected into the right armpit of each mice. Twenty-eight days later, the mice were sacrificed and the cancer tissues were harvested. The tumor volume was calculated every week with the formula (mm3): tumor volume = length × width2 × 0.5. The body weight of the mice was measured by using electronic scale every 7 days. The levels of GAS5 and miR-23a-3p in tumors were detected using qPCR and the protein level of PTEN was analyzed using western blot.
Statistical Analysis
Data are reported as the mean ± SEM and processed with SPSS version 22.0 software (IBM Corp., Armonk, NY, USA). T-test was used to compare the differences between the two groups. One-way analysis of variance (ANOVA) was used for the difference analysis of three groups and more than three groups. ** P<0.05 was considered to be statistically significant.