Patient samples
This work was approved by the Ethics Committee of the People’s Hospital of Liaoning Province. Forty-five paired osteosarcoma tumor tissues and adjacent normal tissues were collected from osteosarcoma patient. All participants have signed the written informed consent.
Cell culture and transfection
Human osteosarcoma cell line MG63, 143B, U2OS and Saos2 were purchased from Shanghai Institutes for Biological Sciences Cell Resource Center (China). The cell lines in this study were validated. The MG63 and 143B cell line were cultured in MEM medium (Gibco, USA) containing 10% FBS (Hyclone, USA) plus 1% penicillin and streptomycin (Solarbio, China). The U2OS and Saos2 cells were maintained in McCoy’s 5A medium (Hyclone) added with 10% FBS (Hyclone) plus 1% penicillin and streptomycin (Solarbio). All cells were placed in a 37 °C incubator with 5% CO2. The siRNA targeting circPIP5K1A (si-PIP5K1A), miR-515-5p mimics and inhibitor and the negative controls (NC), andYAP1 overexpression plasmid (pCMV-YAP1) were purchased from RiboBio (China). The MG63 and U2OS cell were seeded in 6-well plates, and cultured overnight to form a 60% monolayer, and transfected by the pCMV-YAP1 or RNA sequences through a lipofectamine 2000 (Invitrogen, USA) following manufactures’ description.
Cell viability
Cell viability of MG63 and 143B was detected by a MTT experiment. In brief, MG63 and 143B cells were transfected with siPIP5K1A or the NC, and seeded into a 96-well plate (5000 cells/well). Next, 10 µL MTT solution (Sigma, 5 mg/mL) was added into each well at indicated time, and incubated for 4 h. After that, the medium was replaced by 150 µL DMSO and the plates were shaken for 10 min in dark. A microplate detector (PerkinElmer, Germany) was used to detect the absorbance values at 490 nm.
Flow cytometry
Flow cytometry was performed to determine cell apoptosis and CD133+CD44+ cell population. MG63 and 143B cells were transfected with siPIP5K1A or the NC for 48 h, and were collected for further staining. To detect apoptotic cells, the collected cells were stained by an Annexin V-FITC/PI detection kit (Beyotime, China) following manufacturer’s instruction. In Brief, the cells were stained by Annexin V and PI at room temperature in dark for 15 min, subsequently washed with PBS and detected in flow cytometer (BD Biosciences, USA). To determine the portion of stem-like cells, MG63 and 143B cells were incubated with FITC conjugated anti-CD133 (Thermo, USA) antibody and PE-conjugated anti-CD44 antibody (Thermo) and the positive staining was detected by flow cytometer.
Transwell assay
Transwell chambers (Corning, USA) were used to measure cell migration and invasion. Briefly, MG63 and 143B cells transfected with siPIP5K1A or NC were suspended in medium containing no FBS, and were plated in the upper chamber. The lower chamber was filled with medium containing 10% FBS. The invaded cells were fixed in 4% PFA after 24-h incubation, stained with crystal violet (0.2%) for 20 min, photographed by a microscope (Leica, Germany) and counted.
Sphere formation
MG63 and 143B cells (500 cells/well) were seeded into a low-attachment culturing 24-well plate (Corning, USA), in a medium mixture which contains DMEM/F12 (Hyclone, USA), methylcellulose (Sigma), B27 (Sigma), bFGF (20 ng/mL, Sigma), and EGF (10 ng/mL, Sigma). After a 10-day culturing in 37 °C incubator, the formed spheres were captured under light microscope (Leica) and counted. The quantification the spheres was analyzed using Image J software.
RNA extraction and quantification
MG63 and 143B cells were subjected to indicated transfection and lysed by a TRIzol reagent (Beyotime) to extract total RNA. The RNAs were reverse-transcribed to cDNAs by using a cDNA synthesis kit (TransGen, China). Next, the quantification of genes was conducted with a SYBY Green Mixture kit (Thermo). U6 and β-actin were used as internal control for normalization of YAP1, circPIP5K1A, miR-515-5p, separately. The relative level of genes was calculated via a 2−ΔΔCt method. The primers were listed below:
circ PIP5K1A: sense, 5′-AGATTCCCTAACCTCAACCAGA-3′, antisense, 5′-CGAATGTTCTTGCCACCTGC-3′;
miR-515-5p: sense, 5′-CGGGTTCTCCAAAAGAAAGCA-3′, antisense, 5′-CAGCCACAAAAGAGCACAAT-3′;
YAP1: sense, 5′-CAAGACCCATCGGACTGACAG-3′, antisense, 5′-AGCCATAAGCATCAGCTCATTTT-3′;
U6: sense, 5′-AGGGGCCATCCACAGTCTTC′, antisense, 5’-AACGCTTCACGAATTTGCGT-3′;
β-actin: sense, 5′-TGGGTGTGAACCACGAGAA-3′, anti-sense, 5′-GGCATGGACTGTGGTCATGA-3′;
E-Cadherin: sense, 5′-AAAGGCCCATTTCCTAAAAACCT-3′, antisense, 5′-TGCGTTCTCTATCCAGAGGCT-3′;
Vimentin: sense, 5′-GCTGCGAGAGAAATTGCAGGA-3′, antisense, 5′-CCACTTTCCGTTCAAGGTCAAG-3′;
ALDH1: sense, 5′-ACCTCTCACCGCCCTTTATCT-3′, antisense, 5′-GTGAAGGCGATCTTGTTGATCT-3′;
Nanog: sense, 5′-TTTGTGGGCCTGAAGAAAACT-3′, antisense, 5′-AGGGCTGTCCTGAATAAGCAG-3′.
Western blotting
MG63 and 143B cells were subjected to indicated transfection and lysed by RIPA lysis solution (SolarBio) with cocktail of protease inhibitors (Sigma) to extract proteins. A total of 35 μg proteins was resolved in SDS-PAGE, shifted to PVDF membranes, and soaked in 5% nonfat milk for 1.5 h. The membranes were incubated with specific primary antibodies against E-Cadherin (1:1000, Abcam, USA), Vimentin (1:1000, Abcam), β-actin (1:1000, Abcam), ALDH1 (1:1000, Abcam), Nanog (1:1000, Abcam), separately, overnight at 4 °C. Subsequently, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit (1:2000, Abcam) for 45 min. The visualization of bands was achieved by using an ECL solution (Millipore, USA).
Luciferase reporter gene assay
The binding site prediction was performed by an online tool TargetScan (http://www.targetscan.org/vert_71/). The 3′UTR region of YAP1 and circPIP5K1A were synthesized and cloned into pmirGLO vector (Promega, USA) to obtain YAP1-WT and PIP5K1A-WT. Similarly, the mutated luciferase reporter vectors of circPIP5K1A (PIP5K1A-Mut) and 3′UTR of YAP1 (YAP1-Mut) were obtained by inserting corresponding sequences into pmirGLO vector. MG63 and 143B cells were co-transfected with the WT or Mut vectors with miR-515-5p mimics or NC for 24 h. Subsequently, the cells were lysed and the luciferase activity was detected by a dual-luciferase reporter system (Promega).
Immunofluorescence analysis
Cells were solidified at 4% paraformaldehyde for 30 min, treated with Triton X 100 (0.2%) for 10 min and treated with BSA (2%) for 30 min. The slides were hatched with the primary antibody overnight at 4 °C, then hatched with secondary antibodies (Proteintech, China) for 1 h at 37 °C. The slides were stained with the DAPI (Beyotime, China) for 10 min at 25 °C. The Nikon microscope (Tokyo, Japan) was utilized to analyze the immunofluorescence.
Tumor xenograft
BALB/c nude mice aged five-week were obtained from Charles River Laboratories (China). Osteosarcoma cells were transfected with siPIP5K1A or NC, and subcutaneously injected in the right flank (1 × 106 cells in 100 μL saline). The tumor size was measured at indicated time and calculated by the following formula: 0.5 × length × width2. The experiments were approved by the Animal Care and use Committee of the People’s Hospital of Liaoning Province.
Statistics
Data in this work were shown as mean ± SD and processed by a SPSS software (Version 22,0). Student’s t test or one-way ANOVA method was used for comparison between two or more groups. The differences were considered as statistically significant with P values < 0.05.