Over-expression of hsa_circ_0088214 suppresses tumor progression by inhibiting Akt signaling pathway in osteosarcoma

Cell culture

Human osteosarcoma cells, MG63 and U2OS were obtained from the American Type Culture Collection (ATCC, USA). All the cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, USA). All the cells were cultured in an incubator at 37 °C with 5% CO2.

Cell counting kit-8 assay

Three thousand cells were seeded into 96-well plates and cultured overnight. Absorbance at 450 nm (OD450) was recorded at the 1 day, 2 day, 3 day, 4 day and 5 day time points respectively according to the instructions of the Cell counting kit-8 (CCK-8 kit; Beyotime, China). For drug resistance assay, 15,000 cells were treated with different concentration of cisplatin for 24 h. Finally, viability curves were plotted.

Wound healing assay

Osteosarcoma cells were cultured in 6-well plates, and cell monolayer was subsequently scratched with a 200 µL pipette tip. Representative images of cell migration were captured 0 and 24 h after scratching.

Invasion assay

Matrigel was dissolved overnight at 4 °C, and diluted with DMEM. 40 μL diluted matrigel was added into the upper chamber and solidified after incubation at 37 °C for 2 h. 20,000 cells with 200 μL DMEM was added into the upper chamber, and 600 μL complete medium was added into the lower chamber. Non-invaded cells were wiped with cotton swabs after culturing for 24 h, and the left cells were fixed with 4% paraformaldehyde for 15 min, stained with 1% crystal violet for 5 min. Images of cell invasion were captured under a light microscope.

qRT-PCR

RNA extraction and quantitative real-time PCR (qRT-PCR) were performed according to the manufacturer’s instructions. The qRT-PCR was conducted using Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, US) and the Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific). Relative gene expression was calculated by the comparative CT method (ΔΔCT), and GAPDH was used as a housekeeping gene.

Western blot

Cells were lysed with RIPA buffer with 1% proteinase and phosphatases inhibitors. The proteins were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% BSA and incubated with primary antibody overnight at 4 °C. The primary antibodies were used as follows: anti-E-cadherin (1:1000; Cell Signaling Technology, USA), anti-Snail (1:1000; Cell Signaling Technology, USA), anti-Bax (1:1000; Cell Signaling Technology, USA), anti-P-Akt (1:1000; Cell Signaling Technology, USA), anti-Akt (1:1000; Cell Signaling Technology, USA) and anti-β-actin (1:1000; Cell Signaling Technology, USA). Then, the membrane was washed with TBST for three times and incubated with the second antibody for 1 h at room temperature. After washing with TBST for three times, the expression of targeted protein was analyzed by using the electrochemiluminescence reagent.

Hoechst 33342 staining

Cells were seed into 6-well plate and cultured to more than 80% confluence. Then cells were treated with 600 μM H2O2 for 12 h to induce apoptosis. After Hoechst 33342 staining, the nucleus of normal cells is ordinarily blue, whereas the nucleus of apoptotic cells will be densely stained, or clumpy densely stained, with a little whitish.

Cell cycle detection

Cells were seed in 6-well plate and treated with vector or circular RNA overexpression plasmid. Propidium iodide (PI) with the concentration of 20 μg/mL was used for DNA staining and cells were detected by flowcytometry and the data was analyzed by Flowjo software.

Apoptosis analysis

Cells were seed in 6-well plate and treated with vector or circular RNA overexpression plasmid. Cells were treated with 600 μM H2O2 for 12 h in order to induce cell apoptosis and stained with Annexin V/PI. Cell apoptosis ratio was detected by flowcytometry.

Bioinformatic analysis

In order to explore the downstream proteins regulated by hsa_circ_0088214, CircInteractome database (https://circinteractome.nia.nih.gov/) was used for predicting. And the classic protein interaction database STRING (https://cn.string-db.org/) was used to show the interaction of downstream proteins.

Statistical analysis

All data were presented as the mean ± standard deviation (SD). The differences among three groups or more were analyzed by one-way analysis of variance (one-way ANOVA). P < 0.05 was considered to be statistically significant.

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