NGF, MMP-2, FAK, c-Src and β-actin antibodies were obtained from GeneTex International Corporation (Hsinchu City, Taiwan). The phosphorylated forms of FAK (p-FAK) and c-Src (p-c-Src) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). MMP-2, FAK, c-Src and control ON-TARGETplus siRNAs were obtained from Dharmacon (Lafayette, CO, USA). Quantitative polymerase chain reaction (qPCR) primers and probes, as well as Taqman® One-Step PCR Master Mix, were supplied by Applied Biosystems (Foster City, CA, USA). Recombinant human NGF was obtained from PerpoTech (Rocky Hill, NJ, USA). All other chemicals used in this study were supplied by Sigma-Aldrich (St. Louis, MO, USA).
The human chondrosarcoma cell line JJ012 was kindly provided by Dr. Sean P. Scully (University of Miami School of Medicine, Miami, FL, USA), while the SW1353 chondrosarcoma cell line was obtained from the American Type Cell Culture Collection (Manassas, VA, USA). JJ012 cells stably expressing the NGF complementary DNA (cDNA) clone (JJ012/NGF cells) were established according to our previous method (23). Cells were cultured 50%/50% in Dulbecco's Modified Eagle Medium (DMEM)/alpha-minimum essential medium (α-MEM) medium, 10% fetal bovine serum (FBS) and antibiotics, then maintained in a humidified incubator at 37°C in 5% CO2.
Cell migration assay
Chondrosarcoma cells were seeded into the upper chamber of Transwell plates (Costar, NY, USA), while NGF and pharmaceutical inhibitors were added to the lower chamber. After 18 h of incubation, migrated cells were fixed with 3.7% formaldehyde and stained with crystal violet, then counted manually under the microscope (24, 25).
Wound healing assay
The confluent chondrosarcoma monolayer was scratched by a fine pipette tip to create extended scratches in each well. Cells were then treated with the conditions as indicated, migratory activity was evaluated by microscopy after 24 h and the rate of wound closure was quantified (26).
Western blot analysis
After the indicated treatments, chondrosarcoma cells were lysed in RIPA buffer. The extracted proteins were resolved by SDS-PAGE and transferred to Immobilon® polyvinylidene fluoride (PVDF) membranes. Western blot analysis was performed using the methodology described in our previous reports (27-29).
mRNA and miRNA quantification
Total RNA was extracted from chondrosarcoma cells using TRIzol reagent and RNA concentrations were determined using a NanoVue Plus spectrophotometer (GE Healthcare Life Sciences; Pittsburgh, PA, USA). The M-MLV RT kit (Thermo Fisher Scientific; Waltham, MA, USA) and the Mir-X™ miRNA First-Strand Synthesis kit (Clontech; Mountain View, CA, USA) were used to perform reverse transcription of total RNA into cDNA. Quantitative real-time PCR (qPCR) analysis was performed according to our previous reports (30, 31).
The Human MMP-2 luciferase reporter plasmids containing wild-type or mutant sequences of the three prime untranslated region (3’-UTR) encompassing miR-423-5p binding sites were obtained from MDBio, Inc. (Taipei, Taiwan). Chondrosarcoma cells were transfected with the plasmids using Lipofectamine 2000 (Thermo Fisher Scientific; Waltham, MA, USA), then stimulated with NGF for 24 h. Luciferase activity was monitored using a luciferase assay kit (30, 32, 33).
Tumor xenograft study
Four-week-old male BALB/c nude mice (8 in each group) were bought from Taipei’s National Laboratory Animal Center and orthotopically injected with JJ012 or JJ012/NGF cells (5 × 106, resuspended in 100 μL of medium containing 50% serum-free DMEM/α-MEM and 50% Matrigel), according to a previous protocol (23). Tumor growth in the tibiae was monitored each week by bioluminescence imaging using a Xenogen IVIS imaging system 200 (PerkinElmer, MA, USA). At 12 weeks, the mice were euthanized by CO2 inhalation. The lungs were removed and fixed in 10% formalin for further analysis. All animal experiments satisfied the protocols specified by China Medical University’s Institutional Animal Care and Use Committee (IACUC Approval No. 104-154-N).
Immunohistochemistry (IHC) staining
Mouse lung tissues or specimens from a human chondrosarcoma tissue array (Biomax; Rockville, MD, USA) were rehydrated and incubated with primary anti-NGF or MMP-2 antibodies, then treated with biotin-labeled secondary antibody. Finally, the slides were detected using the ABC Kit (Vector Laboratories, CA, USA) and photographed using the microscope.
All values are presented as the mean ± standard deviation (SD). Significance testing on the difference between the groups was assessed by the Student’s t-test and considered significant if the p-value was <0.05.