A total of 18 osteosarcoma patients were included into the study. OS tissues and paired adjacent normal tissues were obtained from Jinling Hospital (Nanjing, China) from January 2018 to January 2020. The study was reviewed and approved by the Ethics Committee of Jinling Hospital (Nanjing, China). And all the patients provided informed written consent authorizing the use of specimens for the intended research. All resected specimens were stored at − 80 °C prior to RNA extraction.
Human OS cell lines (MG63,U2OS, HOS and 143B) and human osteoblast cell line (hFOB 1.19) were obtained from the Cell Bank of the Chinese Academy of Sciences(Shanghai, China). All types of cell were cultured in DMEM (Hyclone; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.) , 100 U/ml of penicillin (Life Technologies) and 100 μg/ml streptomycin (Life Technologies) at 37 °C in 5% CO2 and 95% air.
Three different small interfering (si)RNAs against Lnc NDRG1 and miR-96-5p mimics, mimics NC, miR-96-5p inhibitor and inhibitor NC were designed and synthesized by Ribobio(Guangzhou, China), and transfected into MG63 and 143B cells using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were collected 48h after transfection, and the knockdown or over-expression efficiency was detected by reverse transcription-quantitative PCR (RT-qPCR). The lentivirus-containing short hairpin RNA(shRNA) targeting Lnc NDRG1 was purchased from GenePharma(Shanghai, China), the shRNA was transfected into 143B cell line, after transfecting for 48h, the cells were selected with puromycin(2μg/mL) for 2 weeks to construct stable Lnc NDRG1 knock down cell line.
RNA extraction and RT-qPCR
Total RNA was extracted from the cultured cells and tissues using Trizol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instruction. For the quantification of miRNA, 0.2 μg of total RNA obtained from cultured cells or tissues was reverse-transcribed to cDNA using AMV reverse transcriptase (TaKaRa, Dalian, China). For the quantification of lncRNA and mRNA, 1 μg of total RNA was reverse-transcribed to cDNA using Oligo d(T) primer (TaKaRa, Dalian, China), the real-time PCR analyses were performed using SYBR Green dye(Invitrogen) with LightCycler96 System (Roche, IN, USA). All reactions were run in triplicate. After the reactions were complete, the cycle threshold (CT) values were determined with the LightCycler96 software. A comparative CT method was used to compare each condition to the control reactions. In miRNA RT-PCR reaction, U6 was used as an internal control; in lncRNA RT-PCR reaction, GAPDH was used as an internal control, and the relative level was calculated with the equation 2−ΔΔCT.
Nucleus and cytoplasm extraction
Nuclear and cytoplasmic fractions were isolated with the reagents in a PARIS™ kit (AM1556, Thermo Fisher Scientific, Waltham, USA). MG63 and 143B cells were lysed in Cell Fraction Buffer on ice for 10 min. Subsequently, after centrifugation at 500 g for 3 min at 4 °C, the supernatant was collected as the cytoplasmic fraction. Then, the pelleted nuclei were washed with Cell Fraction Buffer and used as the nuclear fraction.
Cell viability assay
The proliferation capability of OS cells (MG63 and 143B) were assessed by cell counting kit-8 assay (CCK-8) according to the manufacturer’s instruction. The transfected cells were seeded into 96-well plates (5 × 10^3 cells/well). At 12, 24, 36, 48 and 60h after transfection, 10 μl of CCK-8 reagent was added to the test well and incubated for 2 h at 37 °C away from light. The absorbance was measured at a wavelength of 450 nm.
5-Ethynyl-2’-deoxyuridine(EdU) assay was conducted with Cell-LightTM EdU Apollo567 kit (Guangzhou Ruibo Biotechnology). The siRNA or mimics or inhibitor were transfected into MG63 and 143B cells. After transfection for 24 hours, MG63 and 143B cells were seeded in 48-well plates(Corning). When the density of cells turned to 80%, reagent A was added to the medium and cells were cultured for another 4 hours. Then, according to the manufacturer's instruction, the cells were stained. When the staining was done, photos were taken under the microscope (EVOS M7000; Thermo Fisher) at 100×.
Wound healing assay
Wound healing assays were performed to evaluate the migration ability of osteosarcoma cells. After transfection for 24h, MG63 and 143B cells were seeded into six-well plates (8 × 10^5 cells/well). After seeding for 48h, pipette tips (200 μl) were used to scrape a straight scratch in the confluent cell layer and then cultured in serum-free medium. After washing the cells with PBS to remove cellular fragments, each wound was imaged at 0 and 24 h under the microscope (EVOS M7000; Thermo Fisher) at 100×. Cell migration was quantified by measuring the relative wound areas with ImageJ.
For the transwell assay, after transfection for 24h, cells suspended in serum-free DMEM medium were seeded into the upper transwell chamber(Corning). In the lower chamber, DMEM containing 20% FBS was filled in the well. After culturing for 36 hours, the filters were fixed in methanol and stained with 0.1% crystal violet. The upper cells of the filters were gently abraded, and the lower cells migrated across the filters were imaged and counted under the microscope(EVOS M7000; Thermo Fisher) at 100×. The numbers of migrated cells were counted and calculated by ImageJ (NIH).
Luciferase reporter assay
Wild-type and mutant Lnc NDRG1 fragments were constructed and inserted into the luciferase reporter plasmid(GenePharma, Shanghai, China). Osteosarcoma cells were seeded in 24-well plates and co-transfected with 0.2 μg of firefly luciferase reporter plasmid, 0.2 μg of β-galactosidase (β-gal) expression plasmid (Ambion), and equal amounts (50 pmol) of miR-96-5p mimics, miR-96-5p inhibitor or the negative control RNAs with Lipofectamine 3000 (Invitrogen). The β-gal plasmid was used as the transfection efficiency control. The cells were assayed using a luciferase assay kit 24 h post-transfection (Promega, Madison, WI, USA).
The total proteins were extracted through radio-immunoprecipitation assay(RIPA) (Beyotime, Shanghai, China) supplemented with PMSF. The extracted protein lysates were separated in 10% SDS-polyacryl-amide gel electrophoresis(SDS-PAGE) and transferred to 0.22 μm PVDF membranes (Millipore, Massachusetts, USA). The membrane was sealed with 5% skim milk and then incubated with primary antibodies at 4°C overnight. Then the membrane was washed 3 times with 1× TBST for 20 min each. Secondary antibodies were incubated for 1 h at room temperature followed by another 3 times of 10-min wash with 1× TBST. After that, the membrane was incubated with ECL substrate (Thermo Fisher, CA, USA) according to the manufacturer’s instructions and the bands were detected with the SuperSignal West Pico chemiluminescence substrate (Pierce, Thermo Scientific). The protein bands were analyzed with ImageJ.
Male BALB/c nude mice(4 weeks old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China), and randomly divided into two groups (n=5, respectively). 143B cells transfected with a lentiviral vector sh-Lnc NDRG1 or sh-NC were subcutaneously injected into armpit of nude mice (1 × 107 cells/mice), respectively. The volume of the tumors was measured every week after implantation. The tumor volume was calculated according to the formula: tumor volume [mm3 ] = (length [mm]) × (width [mm])2 × 0.52. The mice were sacrificed after 28 days. The xenograft tumors were excised and weighed. Part of the tumors were used for total RNA extraction, and the remains were fixed in 4% paraformaldehyde for 24 h and then processed for hematoxylin and eosin (H&E) staining as well as immunohistochemical staining for Ki67. For the metastasis assay, 143B cells (1 × 10^5) were injected into the tail veins of mice (three mice per group). Lung metastasis was monitored with a Xenogen IVIS Spectrum Imaging System (PerkinElmer, USA). After 8 weeks, the lungs of mice were excised under anaesthesia, and the number of lung metastatic nodules were counted and validated using haematoxylin and eosin (HE)-stained sections by microscopy.
Immunohistochemistry (IHC) was performed according to the manufacturer’s constructions. After the samples were deparaffinized with xylene and rehydrated with ethanol, the samples were incubated with 3% H2O2 for 5 min to block endogenous peroxidase activity. Then, antigen retrieval was performed by incubating the samples with sodium citrate buffer (pH 6.0) for 20 min at 95 °C, after which the samples were blocked with 5% normal goat serum for 10 min at 20 °C. Subsequently, the sections were incubated with polyclonal antibodies against Ki67(1:500, Servicebio, Wuhan, China) at 4 °C overnight and then incubated with secondary antibodies(1:200, Servicebio, Wuhan, China). The image were captured by Olympus FSX100 microscope (Olympus,Japan).
All date were expressed as mean ± SD. Statistical analyses were performed using Prism software (GraphPad Software 8), and consisted of analysis of variance followed by Student’s t-test when comparing two experimental groups. One-way ANOVA analysis was used to compare the differences among groups. Overall survival (OS) rates were determined using the Kaplan-Meier method. All experiments were triplicated, and p < 0.05 was considered significant.