Ethics statement
All patients provided written informed consent and this study was approved by the Medical Ethics Committee of Ningbo Sixth Hospital.
Bioinformatics
The distribution of lncRNA NEAT1 in OS and normal cells was analyzed by TCGA and GTEX database (https://xenabrowser.net/heatmap/). The location of lncRNA NEAT1 was predicted by http://lncatlas.crg.eu/. In http://starbase.sysu.edu.cn/, we searched for the potential target miRNA of lncRNA NEAT1 and the potential target gene of the miRNA.
Clinical tissue specimens
A total of 10 cases of primary OS tissues and the adjacent normal muscular tissues were collected from OS patients who underwent surgical resection at Ningbo Sixth Hospital between February 2019 and November 2019. All tissue specimens were immediately frozen in liquid nitrogen after resection and stored at -80 °C. The patients aged from 12 to 33 years with the median age of 23 years. All patients were diagnosed by laboratory tests and imaging examinations with complete imaging data and determinable stage of OS. No patients have been received chemotherapy or radiotherapy or any other surgeries prior to surgery. Besides, patients with other tumors were excluded. According to the common stages of OS, there were 2 cases in stage Ⅱ A (G2T1M0), 3 cases in stage Ⅱ B (G2T2M0), 4 cases in stage Ⅲ A (G2T1M1) and 1 case in stage Ⅲ B (G2T2M1).
Cell lines and transfection
The osteoblastic cell line hFOB and two human OS cell lines MG63 and U2OS were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultivated in RPMI-1640 medium containing 10% fetal bovine serum (FBS) (Gibco, Carlsbad, California, USA) at 37 °C under an atmosphere of 5% CO2/95% air.
Subsequently, the lncRNA NEAT1 interference plasmids (sh-NEAT 1,2,3#) and corresponding empty plasmids (sh-negative control [NC]), mimic/inhibitor and mimic/inhibitor control of miR-579 (Thermo Fisher Scientific Inc., Waltham, MA, USA) were transfected into the well-developed MG-63 and U2OS cells. All operations of the transfection were carried out in strict accordance with the manufacturer’s protocol of Lipofectamine 2000 Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Forty-eight h after transfection, the cells were collected for follow-up experiments, and the expression level of corresponding genes was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) to evaluate the transfection efficiency.
RT-qPCR
Trizol regent (Takara Bio Inc., Otsu, Shiga, Japan) was used for total RNA extraction from cultured cells. Then, the SYBR Green qPCR Mix Kit and the ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA) were used to determine mRNA levels. All primers were purchased from Invitrogen (Shanghai, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were used for endogenous control as appropriate. Primer sequences are listed below.
miR-579 Forward 5’-CGTGCCGTTCATTTGGTATAAAC-3’,
Reverse 5’-GAGCAGGGTCCGAGGT-3’;
LncRNA NEAT1 Forward 5’-TGAGTAGTGGAAGCAGGAGGA-3’,
Reverse 5’-GGAGGCAAGGACGAGACAGA-3’;
MMP13 Forward 5′-GACTTCCCAGGAATTGGTGA-3′,
Reverse 5′-TGACGCGAACAATACGGTTA-3′;
GAPDH Forward 5’-GAGTCCACTGGCGTCTTCAC-3’,
Reverse 5’-ATCTTGAGGCTGTTGTCATACTTCT-3’;
U6 Forward 5’-CTCGCTTCGGCAGCACA-3’,
Reverse 5’-AACGCTTCACGAATTTGCGT-3’.
The relative quantitative expression of interest genes was expressed as fold change (2-ΔΔCt method). ∆∆Ct = [Ct (target gene) - Ct (GAPDH/U6)] experimental group - [Ct (target gene) – Ct (GAPDH/U6)] control group. Each sample was examined in triplicate.
Transwell assay
Cell invasion was detected in a Nunc™ polycarbonate inserted cell culture device (Thermo Fisher Scientific Inc., Waltham, MA, USA) pre-positioned with 8-µm perforated plate. In brief, cell suspension was seeded in the apical chamber, and the chemokine was added to the basolateral chamber. After 24 h of routine culturing, cells were stained with crystal violet and photographed under an optical microscope. The indirect counting method was used to count cells.
For migration detection, we only need to remove the matrix gel, the procedure is the same as the invasion experiment.
Subcellular localization
First, we used Lncatlas (http://lncatlas.crg.eu/) to predict the position of lncRNA NEAT1. Fluorescence in situ hybridization (FISH) experiment was used to further determine the location of lncRNA NEAT1 in MG-63 and U2OS cells, and the cell solution was treated with trypsin. After denaturing, the cells were hybridized with probes, and the nuclei were stained and photographed under a fluorescence microscope. Finally, the nucleus was isolated from the cytoplasm by the PARIS kit (cat. No. am1921; Thermo Fisher Scientific Inc., Waltham, MA, USA), and the expression level of lncRNA NEAT1 was detected in the nucleus and cytoplasm.
Western blot analysis
The expression level of epithelial-mesenchymal transition (EMT) related markers in OS cells was detected by Western blot analysis. The cell lysate "Western and IP cell lysate" (No.: p0013) purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China) was thawed and mixed up. The equivalent cracking solution was added to phenylmethyl sulfonylfluoride until the concentration was 1 mM (No.: ST506, Beyotime Biotechnology Co., Ltd., Shanghai, China). The transfected cells were lysed with Western and IP cell lysates. After full cracking, the cells were centrifuged at 10000–14000 g for 3–5 min with the supernatant collected. Bicinchoninic acid kit (No.: p0009, Beyotime Biotechnology Co., Ltd., Shanghai, China) was used to determine the protein concentration. Electrophoresis was then performed in polyacrylamide gel (5% concentrate and 12% separation gel). Tris-buffered saline Tween-20 (TBST) containing 5% bovine serum albumin (BSA) was used to seal the membrane in a decolorizing shaker at room temperature for 1 h. The sealing solution was discarded, and the membrane was put into the plastic groove added with 5% BSA to prepare the primary antibody solution of corresponding concentration overnight at 4 °C. The membrane was washed with TBST three times, 10 min each time. Then the membrane was incubated with the secondary antibody solution at 4 °C for 4 h. The membrane was washed with TBST 3 times, 10 min each time. The membrane was immersed in electrochemiluminescence developer (wbkls0100, Merck Millipore, Billerica, MA, USA) for visualization. The relative optical density (OD) of all immunoblotting bands was analyzed. The antibodies (Abcam Inc., Cambridge, MA, USA) included primary antibodies E-cadherin (1:30000, ab40772), N-cadherin antibody (1:100, ab18203), Vimentin (1:3000, ab92547), GAPDH (1:2500, ab9485) and corresponding horseradish peroxidase labeled secondary antibody (1:50000, ab205718).
Dual luciferase reporter gene assay
The relationship among lncRNA NEAT1, miR-579 and MMP13 was detected by dual luciferase reporter gene assay. The wild type (WT)/mutant type (MUT)-lncRNA NEAT1 and WT/MUT-MMP13 3'UTR including the binding site regarding miR-579 were synthetized by GenePharma (Shanghai, China) and subcloned to pMIR-REPORTTM vector (Thermo Fisher Scientific Inc., Waltham, MA, USA) [26]. The activity intensity of luciferase was detected by Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA).
Cell counting kit-8 (CCK-8) assay
After the cells were treated with 10 µL/well CCK-8 solution (Beyotime Biotechnology Co., Ltd., Shanghai, China), the cells were cultured at 37℃ for 2 h, and then the OD at 490 nm was measured with BioTek instruments.
Colony formation assay
The transfected cells were treated with trypsin and then collected and added to the 6-well plate (200 cells/well). The cells were cultured in Dulbecco’s modified Eagles Medium containing 10% FBS for 2 weeks under the external environment of 37℃ and 5% CO2. The medium was changed regularly once every 3 days. Cells were fixed with methanol for 15 min at room temperature, stained with 1% crystal violet for 30 min at room temperature, and images (including > 50 cells/colony) were captured with an optical microscope (magnification, × 100; Olympus Optical Co., Ltd, Tokyo, Japan). Finally, the number of visible colonies was counted manually.
Statistical analysis
Data were analyzed using SPSS 21.0 (IBM Corp. Armonk, NY, USA). Data were in normal distribution according to Kolmogorov-Smirnov method and described as mean ± standard deviation. Differences among multiple groups were analyzed using one-way analysis of variance (ANOVA) or two-way ANOVA. Tukey’s multiple comparisons test was used for the pairwise comparison after ANOVA analysis. p was obtained by two-tailed test and p < 0.05 was considered statistically significant.