Clinical tissue collection
OS tissues and adjacent normal tissues were collected from 35 patients at the First Affiliated Hospital of Jinzhou Medical University (Liaoning Province, China) between 2016 and 2018, which were immediately frozen in liquid nitrogen for subsequent quantitative real time PCR analysis. Meanwhile, the corresponding basic clinicopathological characteristics were recorded. Before surgery, all the patients have not received any radiotherapy or chemotherapy. All participants signed the written informed consent and this work obtained the approval from the Ethics Committee of First Affiliated Hospital of Jinzhou Medical University (Approval number: JM3643; 2017.7.23).
Cell culture conditions
Human OS cell lines, including U2OS, MG-63 and Saos-2, as well as osteoblast cell line (hFOB1.19) were obtained from Shanghai Institute for Biological Sciences (Shanghai, China), which were all cultured in DMEM (Solarbio, Beijing, China) supplemented with 10% FBS (Beyotime, China) and maintained at 37 °C in a humidified incubator containing 5% CO2.
MiR-20b-5p mimics and miRNA negative control (miR-NC), as well as small interfering RNA targeting KIF23 (si-KIF23) and si-NC were provided by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The empty vector (pcDNA3.1) and the plasmids containing KIF23 were purchased from GenePharma Co., Ltd. (Shanghai, China). For miR-20b-5p overexpression, miR-20b-5p mimics or miR-NC at a final concentration of 50 nM was transfected into MG-63 or U2OS cells seeded in a 6‑well plate at a density of 5 × 105 cells per well. For KIF23 knockdown, si-KIF23 or si-NC was transfected into U2OS cells (5 × 105 cells per well). In the rescue experiments, co-transfection of miR-20b-5p mimics and KIF23 was performed in U2OS cells. The above cell transfection was conducted using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, transfected cells were harvested for further experiments.
Quantitative real time PCR
Tissue samples and cells were pre-treated with an RNA extraction kit (Takara, Beijing, China) to isolate total RNA. Total 2 µg of total RNA was used to synthesize the cDNA using an ABScript II cDNA First Strand Synthesis kit (Invitrogen). Quantitative real time PCR analysis was performed using A SYBR Premix Ex Taq™ Real-Time PCR Kit (Thermo Fisher Scientific, Inc.) on an ABI PRISM 7500 sequence detection system. The relative expression of miR-20b-5p and KIF23 was calculated by the 2-ΔΔCq method with U6 and GAPDH as the internal controls, respectively.
Cell proliferation assay
The MG-63 or U2OS cells at a density of 3,000 cells per well were seeded in 96-well plates and cultured in complete media overnight. At 24, 48 and 72 h after seeding, cells were incubated with 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) at 37 °C for 2 h. Subsequently, the absorbance value was measured at 450 nm at the indicated time points using a microplate reader.
Flow cytometry analysis
For cell cycle analysis, transfected cells were seeded on 6-cm dishes (1 × 105 cells/dish) and collected until 80% confluence. Then, the cells were fixed with 0.8 ml of 70% ethanol for 30 min at 4 °C. After centrifugation, we discarded the ethanol and stained the cells with a propidium iodide (PI, 100 μg/ml) solution containing 10 μg/ml of DNase-free RNase A for 30 min at room temperature. Afterwards, stained cells were analyzed using flow cytometer (Cell Lab Quanta, Beckman Coulter). Similarly, apoptosis was detected by Annexin V-FITC/PI apoptosis detection kit (KeyGEN Biotech, Nanjing, China) following manufacturer’s instructions.
Transwell migration assay
The migration ability of MG-63 or U2OS cells was evaluated by transwell assay using Transwell chambers (BD Biosciences, CA, USA). Approximately 5× 104 transfected cells were added to the upper chamber after suspended in 200 μL FBS-free DMEM medium. At the same time, FBS dissolved in 500 μl of culture medium was added to the lower chamber. Being cultivated for 24 h, the cells that migrated on the lower chamber were stained with 0.1% crystal violet. Five randomly fields were selected to count the number of migrated cells was counted using a light microscope.
Luciferase reporter assay
The wild-type (WT) and corresponding mutant (MUT) 3’-UTR of KIF23 in the miR-20b-5p binding sites was amplified by Shanghai GenePharma Co., Ltd. Then, the amplified WT KIF23 and MUT KIF23 were respectively inserted into the psiCHECK-2 vector (Promega Corporation, Madison, WI, USA). Subsequently, the chemically synthesized reporter vectors were co-transfected with miR-20b-5p mimics or miR-NC into MG-63 and U2OS cells using Lipofectamine 2000 reagent. The Dual Luciferase Reporter Assay kit (Promega Corporation) was utilized to analyze the relative luciferase activities after 48 h transfection.
Western blot analysis
RIPA lysis buffer (Beyotime, China) and enhanced BCA Protein Assay Kit were used to extract total protein and quantify protein concentration, respectively. After separated by 10% SDS-PAGE, the protein was transferred onto PVDF membranes (EMD Millipore). Following blocked with 5% fat-free milk prepared in TBST, the membranes were incubated with primary antibodies against KIF23, CDK4, Cyclin D1, Bad, Bcl-2, PCNA, Ki-67, E-cadherin, N-cadherin and GAPDH (all from Abcam, Cambridge, UK) overnight at 4 °C. After washing twice with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature, followed by protein detection with an enhanced chemiluminescence kit (Beyotime, China).
The data were expressed as mean ± standard deviation (SD). Spearman’s correlation analysis was conducted to assess the correlation between miR-20b-5p and KIF23. The correlation between miR-20b-5p or KIF23 expression and the clinicopathological characteristics of OS was evaluated using Chi-square test. The differences between two groups was evaluated by Student’s t-test and differences among more than two groups were assessed by one-way analysis of variance (ANOVA) followed by Dunnett’s t-test. The statistically significant differences were accepted if a value of p less than 0.05.