Clinical tissue specimens
Tissue specimens were obtained from the tumor tissues of 6 cases of OS patients and 3 cases of normal bone tissues obtained during the diagnosis and treatment of the Orthopedics Department of the Second Hospital of Shanxi Medical University (Taiyuan, China), and then qRT-PCR and Western blot were carried out. In the pathology department, 30 paraffin-embedded specimens of osteosarcoma patients and 6 paratumoral bone tissue specimens were selected for immunohistochemistry. All patients obtained informed consent. The research protocols were approved by the Ethics Committee of the Second Hospital of Shanxi Medical University, (2021) YX No. (098).
Cell line and culture
The human osteosarcoma cell lines Saos-2, U2OS, Mg63 and human chondrogenic cell line C28 were used in this work. Saos-2, U2OS, Mg63and C28 cells were incubated in complete culture medium with supplementary 10% fetal bovine serum, 1% penicillin and 89% DMEM/F-12 (Gibco, USA), and placed in a 37-degree cell incubator with 95%humidity and 5% CO2.
RNA extraction and qRT-PCR
Total RNA was extracted used TRIzol reagent (Thermo Fisher Scientific). PrimeScript RT Master Mix kit (Takara) was applied to reverse transcription. Amplify cDNA we use SYBR Green Premix Ex TaqII kit (Takara, Japan). Use the QuantStudio™ 6 Flex real-time fluorescence quantification system (Thermo Fisher Scientific) to evaluate the relative expression and the internal reference 18S. The sequences of NDN are 5'-CGAGCTCATGTGGTACGT-3' and 3'-CGATGACATCTTTCACCATGTC-5'. The sequences of 18S are 5'-AAACGGCTACCACATCCA-3' and 3'-CACCAGACTTGCCCCTCCA-5'. The sequence of VEGFA is 5'-GTTTGACAAGACCACCAAACT-3' and 3'-CCGCATAATCTGCATGGTGAT -5'. The qPCR reaction conditions for NDN, VEGFA, 18S are 95℃ 15 min, 95℃ 30 s, 55℃ 5 s, 72℃ 5 s, A total of 40 cycles.
Tumor tissue, normal bone tissue adjacent to the tumor, and xenograft tumor tissue of patients with osteosarcoma were fixed (4% paraformaldehyde), dehydrated, embedded in paraffin, sliced (5mm), then deparaffinization, rehydration and other standard immunohistochemical staining procedure were performed. The sections were incubated with anti-NDN/VEGFA/CD31 (Santa Cruz Biotechnology, Dallas, TX, USA) primary antibody one night at 4°C. After washed, biotin-labeled goat anti-mouse/rabbit IgG (PV-9000, Beijing Zhongshan Bridge) was incubated at 37°C for 1 h, and then stained with hematoxylin. A slide scanner and software (purchased from 3DHISTECH, Hungary) were used for image acquisition, and ImageJ image analysis system was used for positive result determination.
Saos-2 and U2OS cells were cultured in a six-well plate (1x105 cells per well). When the cells reached 30% confluence, use lentivirus UP-NDN (LV-NDN, Genechem, Shanghai, China) and negative control lentiviruses (LV-Control) were transfected into Saos-2 at MOI = 5, and U2OS cells at MOI = 10 for 16 hours.
Western blot analysis
The levels of target protein were analyzed by western blot and H2b and orglyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression act as the control. Cells and tissue were disintegrated in RIPA buffer containing PMSF, phosphatase and protease inhibitors (Keygen, Nanjing, China) on ice. Sample were separated in 10% SDS/PAGE gels, then transferred to nitromethyl cellulose membra ((BOSTER, Wuhan, China). The membranes were probed with anti-NDN (FLT-1; sc-101224,1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), anti-VEGF (sc-7269,1:500 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), anti-H2B (Abcam USA ab6877). GAPDH (cat. 5174, 1:800 dilution; Cell Signaling Technology). ECL kit (BOSTER, Wuhan China) were utilized to develop the signals. The quantification of protein levels in the tumor were carried out via normalizing with H2B and GAPDH by IMAGEJ software.
Transwell invasion assay
Matrigel Basement Membrane Matrix (Cat.354248, Corning, England) was diluted with DMEM/F12 without FBS at a dilution of 1:7 and was added to the upper Transwell chambers 70µl/well. After incubated in 37℃ for 2 hours, each upper chamber was added with 40000 cells suspended in 150 µL serum-free medium, while 650µl complete medium for each lower chamber. After 36 h, the cells on the upper surface are erased, and the cells on the lower surface was dyed with 1% crystal violet for 30 min, photographed under a microscope.
Apoptosis detection (flow cytometry)
Take the transfected Saos-2 and U2OS cells in the logarithmic growth phase, digest the cells with 0.25% trypsin without EDTA, wash the cells twice with PBS (centrifuged at 1000 rpm for 5 min), and take 100 µl containing 5×105 Add 5µl Annexin V-PE (4A Biotech, China) to the cell suspension in a flow tube. After mixing, incubate for 5 minutes in the dark, add 400µl PBS, and then add 10µl Annexin V-7AAD by pipetting and mixing, and immediately perform streaming detection.
After mixing Matrigel (Cat.354248, Corning, England) with DMEM/F-12 serum-free medium at a ratio of 1:1, add 50µl of diluted Matrigel to each well of a 96-well plate. When the Matrigel was solidified, add 1×104 cells/100µl HUVEC cells per well to 96-well plate, and add 100µl culture supernatant of saos-2 cells transfected with LV-NDN and LV-Control, and incubate for 24h. A positive control group was set up, and 10 ng/mL human VEGF 165 recombinant protein was added to each well. Observe the angiogenesis in the hole under a microscope, and use Image J software to calculate the total length of blood vessel branches in each field of view.
Mouse model and bioimaging analysis
Animal welfare and experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006), and approved by the animal ethics committee of Shanxi Medical University. BALB/c female nude mice aged 4 to 6 weeks were randomized and implanted subcutaneously with 1X106 Saos-2/LV-UP-NDN and Saos-2/LV-CON-NDN cells in 200 µl Matrigel (Cat.354248, Corning, England) in their back (n = 6 per group). The implanted tumors were monitored weekly. After inoculation, individual tumor-bearing mice were anesthetized by being given an i.p. injection of ketamine (75 mg.kg-1) and dexmedetomidine (0.5 mg.kg-1) after being injected i.v. with 2nM of MMPSenseTM680 for 24 h. Then bioimaging was conducted and the vascularization was determined by FMT using TRUEQUANT software (Perkin Elmer, Waltham, MA, USA). The tumors were dissected to analyze their volumes and weights after experiment. Tumor tissues were saved in 4% paraformaldehyde, or RNAlater solution or lysis buffer.
In this experiment, FLAG+ cell samples were used as the control group, and FLAG+Ndn as the experimental group. Flag-Necdin (bait protein) COIP was performed with flag magnetic beads. In the flag antibody test results, no bait protein was detected in the control group. The bait protein can be detected in the experimental group, indicating that the COIP experiment was successful.