Tissue samples
This study was approved by the ethics committee of the Second Affiliated Hospital of Southern Medical University. All human osteosarcoma and para-carcinoma samples were obtained from a total of 25 patients undergoing biopsies before receiving chemotherapy and radiotherapy at the department of joint and orthopedics. Tissue samples obtained from biopsy were collected and instantly frozen in liquid nitrogen. The pathological and personal clinical information is listed in Table 1.
Obtainment and analysis of original data
GES65071 from the GEO database was downloaded. The R package affy was used in background correction and normalization processing. The R package limma was used to detect the difference of the miRNA expression level between normal samples and OS samples. The filter criterion: Log [fold change (FC)] > 1 and adj. P value < 0.05.
Cells and cell cuture
All human OS cells, including Saos-2, U-2 OS, MG-63, MNNG, 143B and normal osteoblast cell line hFOB 1.19 were obtained from the American Type Culture Collection (ATCC, Manassas, US). OS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, US) replenished with 10% fetal bovine serum (FBS) (Gibco, NY) and 1% penicillin/streptomycin (PS, Gibco, CA). The hFOB 1.19 cells were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/F12) (Life Technologies, NY) with 0.3mgl/mL of G418, 10% FBS and 1% PS. All cells were incubated with 5% CO2 at 37℃.
Establishment of transfected cells
Plasmids overexpressed miR-744-5p and TGFB1 were used in the vitro experiments. The cells were cultured in 6-well plates. After washing with DMEM, the complex liquid of transfection was added into the plates and incubated for 24 h. Then the cells were cultured with DMEM containing 10% FBS for 48 h. G418 selective media was used to screen out the transfected cells. The lentiviral transfection was conducted in vivo experiments. The cells were cultured in 24-well plates for 24 h. The medium with 2 µg/ml polybrene was used to replace the original medium, and lentivirus transfected with miR-744-5p or TGFB1 were added into the wells. After incubating for 24 h, the cells were cultured with DMEM for another 72 h. The transfection efficiency was examined via qRT-PCR .
Quantitative real-time PCR (qRT-PCR)
Total RNAs from cells and tissue samples in Trizol (Invitrogen, US) were extracted from the frozen pulverized samples according to the manufacturer’s protocol. 500 ng of total RNA was reverse transcribed into cDNA. The cDNA was diluted five times with enzyme-free water. One-step qRT-PCR was performed in a 10 µL reaction system. The purity and integrity of the total RNAs were examined through the absordance at 260nm and 280 nm with. The primers of TGFB1, U6 and GAPDH were purchased from TsingKe (Beijing, China) and the primers of miR-744-5p were designed personally based on the purchased primers. Reverse transcription (RT) was performed with SuperScriptTM Preamplification System for First Strand cDNA Synthesis according to the protocol of manufacture, and qPCR was performed with LightCycler® Real Time PCR. The expression of U6 or GAPDH served as endogenous control. The sequences of the primers are as follows: TGFB1 forward: 5’-GGCCAGATCCTGTCCAAGC-3’; TGFB1 reverse: 5’-GTGGGTTTCCACCATTAGCAC-3’; GADPH forward: 5’-GGAGCGAGATCCCTCCAAAAT-3’; GAPDH reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’; U6 forward: 5’-CTCGCTTCGGCAGCACA-3’; U6 reverse: 5’-AACGCTTCACGAATTTGCGT-3’; miR-744-5p forward: 5’-AATGCGGGGCTAGGGCTA-3’; miR-744-5p reverse: 5’-GTGCAGGGTCCGAGGT-3’.
Western Blotting (WB)
Proteins of the cells were extracted, and the concentration of proteins was assessed with the BCA protein assay kit (Beyotime, China). Then the proteins were electrophoresed through 10% SDS-PAGE for 4 h at 40 volts and then transferred to the PVDF membranes. The proteins were incubated with specific primary antibodies at 4℃ overnight. After washing with TBST, the proteins were incubated with the secondary antibodies at indoor temperature for 2 h. Rat anti-TGFB1 (1:1000, Abcam), GAPDH (1: 10,000, Proteintech), N-cadherin (1:1000, Abcam), E-cadherin (1:1000, Abcam), Vimentin (1:1000, Abcam), p-P38 (1:1000, Abcam), t-P38 (1:1000, Abcam), antibodies were used to detect the proteins. Reacting bands were acquired with ECL reagent and the quantity analysis was performed with ImageJ normalized to GAPDH.
Colony formation assay
About 800 OS cells were seeded into the six-well plate and cultured with DMEM and 10% FBS at 37℃ for 1 week. When the colonies turned invisible they were washed with PBS and fixed with 4% paraformaldehyde, then satined with 0.1% crystal violet. The images were captured with a scanner and the counts were calculated manually.
5-Ethynyl-2-Deoxyuridine (EdU) Incorporation assay
The EdU incorporation assays were performed in accordance with the manufacturer’s protocol. 1 * 104 cells/well of OS cells were seeded into 96-well plate and cultured with 100 µl of 50 µM EdU medium for 2 h. Then the cells were fixed with 4% paraformaldehyde and destained with 2mg/mL glycine. Next, Apollo staining was performed with 1X Apollo dyeing reaction fluid. 1X Hoechst 33342 reaction mixture was used in DNA staining. At least 50 cells per well were selected randomly. The intensity was measured from five random fields and the photos were taken with fluorescent microscope (Carl Zeiss, Germany).
Transwell Migration and invasion assay
Transwell migration assay was performed to detect the cell migratory ability. Total of 4.0×104 cells were seeded in the upper chamber with 200 µL of DMEM, while the lower chamber was immersed with 600 µL of DMEM with 10% FBS. After incubating for 24 h, the lower chamber was moved away and cells were fixed with 4% paraformaldehyde for 30 min. Then stained the cells with 0.1% crystal violet for 20 min, unmigratory cells on the upper chamber were wiped with swab. After removing the crystal violet, five randomized fields were observed and photographed with a microscope. As for the Transwell invasion assay, Matrigel (BD 5mg/ml) was diluted to 1mg/ml with serum-free medium. 100 µl of resulting Matrigel was put on the upper chamber and incubated at 37℃ for 1 h. The following steps were the same with the Transwell migration assay.
Luciferase reporter assay
Possible miR-744-5p-binding sites were obtained from the miRDB database. Wild-type TGFB1 (WT-TGFB1-3’-UTR) and mutant TGFB1 (MUT-TGFB1-3’-UTR) were synthesized by GenePharma (Shanghai, China). Cells overexpressing miR-744-5p was transfected with WT-TGFB1-3’-UTR and the negative controls were transfected with MUT-TGFB1-3’-UTR. 48 h later after transfection, the luciferase activity was determined with the Dual-Luciferase Assay System (Promega, WI, US) and normalized using Renilla luciferase.
Immunohistochemistry (IHC)
The slides were immersed in the miscible liquids of potassium dichromate and concentrated sulfuric acid, then flushed for 1 h. Polylysine was smeared on the surface. Tissues were conducted through paraffin embedding. Tissue sections were dewaxed with xylene and ethanol. Then the sections were immersed in 0.01 mol/L sodium citrate buffer for 10 min and 3% hydrogen peroxide for 30 min at indoor temperature. Next, the sections were put into phosphate-buffered saline (PBS) for 5 min, and sealed with 5% bovine serum for 0.5 h at 37℃. The tissue sections were incubated with primary antibodies overnight at 4℃. After washing three times with PBS, the sections were incubated with secondary antibodies for 0.5 h at 37℃ and then incubated with SABC for another 0.5 h. Color developing agents were added after wiping up the sections and then hematoxylin staining was performed. Finally, the sections were dehydrated with ethanol and xelene, and sealed with resinene. The main antiboies were Ki-67, E-cadherin, N-cadherin and Vimentin (Abcam, UK). Photos were captured with an orthophoto microscope.
Hematoxylin-eosin (HE) staining
The tissues were immersed in stationary liquid containing 10% methanal. After dehydrating with ethanol and xylene, the samples were embedded in the paraffin. The sections were dewaxed with xylene and ethanol before staining. The sections were successively immersed in Hematoxylin, hydrochioric acid, ammonium hydroxide and flushed with distilled water for 1 h. Next, the sections were dehydrated in the ethanol and dipped in eosin staining solution. Finally the sections were immersed in ethanol and xylene, and sealed with gums.
Animal experiments
Nude mice in the study were purchased from Animal Core Facility of Southern Medical University and were randomly divided into 5 groups, 5 in each group. OS cells with fluorescent protein RFP were inoculated into subcutaneous tissue of the nude mice. Pulmonary metastasis models were conducted through tail vein injection. The volume and size of tumors were recorded every 3 days and the tumors were seperated and imaged on day 28th. The mice were sacrificed at the end of experiments.
Statistical analysis
All experiments were repeated at least 3 times and data were demonstrated as means ± standard deviation. Independent Students’ t-test and One-way ANOVA were used to compare the difference between two groups in clinical characteristics. Paired t-test was used to evaluate the differences in the miRNA expression between TGFB1 and miR-744-5p in tissue samples. Pearson’s chi-squared test was performed to detect the relation between miR-744-5p and TGFB1. Log-rank test was conducted to evaluate the prognosis and overall survival of OS patients. Statistical analyses were performed with SPSS, v. 23.0. p < 0.05 was considered statistically significant. Data are presented as the means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001