Cell culture
The human osteoblast cell line hFOB1.19 was purchased from the cell bank of typical culture preservation Committee of Chinese Academy of Sciences. The human OS cell line Saos-2 was purchased from Shanghai gaining Biotechnology. The human OS cell lines HOS, MG-63 and U2OS were purchased from American Type Culture Collection (ATCC). The hFOB1.19 cells were maintained in DMEM and Ham’s F12 medium (DMEM/F12, Gibco BRL, USA) (1:1) with 0.3mg/mL G418. HOS cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco BRL, USA). MG63 cells were cultured in RPMI 1640. Saos-2 and U2OS cells were cultured in McCoy’s 5A medium. All cells were supplemented with 10% fetal bovine serum (FBS, Gibco BRL, USA), penicillin (100 U/mL) and streptomycin (100 mg/mL). The hFOB1.19 cells were cultured at in 5% CO2 at 33.5℃, and the other cells were cultured in a humidified atmosphere of 5% CO2 at 37°C.
siRNA and plasmid transfection
The specific small interfering RNA (siRNA) targeting SIRT2 and negative control siRNAs were purchased from GenePharma (China). MG63 and Saos-2 cells were seeded in 6-well plates, and the cells were transfected with 20 nM siRNAs using jetPRIME transfection reagent according to the manufacturer’s instructions. For overexpression experiments, U2OS cells were transfected with SIRT2-expression plasmid penter-SIRT2-C-Flag-His using Lipofectamine 2000.
The siRNA sequences were as follows:
si-SIRT2-1: 5’-CCTAGAGGCCAAGGCTTAAdTdT-3’;
si-SIRT2-2: 5’- GAGGCCAUCUUUGAGAUCAGCUAUU-3’;
si-NC: 5’- UUCUCCGAACGUGUCACGUTT − 3’.
Lentivirus infection
Lentivirus shRNAs (expressing green fluorescent protein, GFP) targeting SIRT2 (shSIRT2) were purchased from GeneCopoeia (China). To acquire a MG63 cell line with SIRT2 stably knockdown (MG63-shSIRT2), shSIRT2 lentivirus were infected into MG63 cells (Multiplicity of infection, MOI = 80) using 5 µg/mL polybrene transfection reagent (GenePharma, China) and cells were selected with 0.5µg /mL puromycin.
Quantitative Reverse-Transcription PCR
RNA was extracted using TRIzol reagent (Invitrogen, USA) and reverse-transcribed to cDNA by the HiScript Q RT SuperMix (Vazyme, China) according to the manufacturer’s instructions. ChamQ SYBR qPCR Master Mix (Vazyme, China) was used to amplify the products and monitored on CFX96 Touch Real-Time PCR detection system (Bio-Rad, USA). PCR primer sequences were as follows:
SIRT2-F: CTGCGGAACTTATTCTCCCAGAC;
SIRT2-R: CCACCAAACAGATGACTCTGCG;
GAPDH-F: GCACCGTCAAGGCTGAGAAC;
GAPDH-R: GCCTTCTCCATGGTGGTGAA.
Western blot
Total cellular proteins were extracted with radio immunoprecipitation assay (RIPA, Beyotime Biotechnology, China) lysis buffer with phenylmethanesulfonyl fluoride (PMSF, Beyotime Biotechnology, China) and protease inhibitor (BestBio, China) at a 100:1:1 ratio. Protein concentrations were measured by the BCA Protein Assay Kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions. Proteins were separated by polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes (Millipore, USA). After 2 h blocking with 5% skimmed milk, the membrane was incubated in primary antibody at 4°C overnight. Specific primary antibodies included: SIRT1 (1:1000, 13161-1-AP, Proteintech), SIRT2 (1:1000, 19655-1-AP, Proteintech), EMT Antibody Sample Kit (1:1000; 9782; Cell Signaling), MMP2 (1:1000, 40994, Cell Signaling), MMP9 (1:1000,13667, Cell Signaling), Snail (1:1000, 13099-1-AP, Proteintech), GAPDH (1:1000, 10494-1-AP, Proteintech), β-tubulin (1:1000; 86298; Cell Signaling), Flag (1:1000; 20543-1-AP; Proteintech) and HA (1:1000; 66006-2-Ig; Proteintech). The following day, membranes were washed 3 times by Tris-buffered saline with Tween 20 (TBST), then incubated with goat anti-mouse HRP-conjugated secondary antibody (ZB-2305) or goat anti-rabbit HRP-conjugated secondary antibody (ZB-2301) (1:2,000) for 1 h. The proteins were visualized with the Immobilon Western Chemiluminescent HRP Substrate kit (Millipore, USA).
Cell viability assay
The cell viability was assessed using the Cell Counting Kit 8 (CCK-8, BestBio, China) according to the manufacturer's protocol. MG63 and Saos-2 cells with SIRT2 knockdown and U2OS cells with SIRT2 overexpression were seeded into a 96-well plate. At 0, 24, 48, 72 and 96 h after seeding, 10 µL CCK-8 reagent was added and incubated for 2 h at 37°C, then the absorbance of the solution was measured at a wavelength of 450 nm.
Wound-healing assay
Cell migration was examined using the wound-healing assay. Briefly, the transfected cells were plated and cultured overnight to about 80–90% confluence in the 6-well plate. A wound was created by scraping a straight scratch in the confluent cell layer with a pipette tip (200 µL). Cells were washed 3 times with PBS to remove the floating cells and serum-free culture medium was added. A computer-based microscopy imaging system was used to capture the image of scratched positions at 0 h and 24 h. The ratio of the area of wound-healing was quantified by Image J software (NIH, Bethesda, MD, USA).
Transwell migration and invasion assay
For migration assay, 8 × 104 MG63 or Saos-2 cells in 200 µL culture medium without FBS were seeded in Transwell chambers and the lower chambers were filled with 500 µL 20% FBS complete medium as a chemoattractant. After incubation for 36 h, cells that have migrated to the lower chamber were washed by PBS, fixed by 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 10 min. The number of migrated cells was counted in five randomly selected fields under phase contrast microscope.
For invasion assay, a layer of artificially reconstituted basement membrane material Matrigel was coated on the bottom of the upper surface of the cell membrane (the dilution ratio of Matrigel to serum-free medium is 1:6).
Immunofluorescence
Transfected cells were seeded in 24-well plate containing slides. When cells were 60% confluent, cell slides were fixed in 4% paraformaldehyde for 15 min, and permeabilized in 0.2% Triton X-100 (Sigma, USA) for 30 min. Cell slides were blocked in 5% BSA for 1 h, then incubated with N-cadherin (1:100, 22018-1-AP, Proteintech) or Vimentin (1:100, 10366-1-AP, Proteintech) antibody at 4°C overnight. The following day, cells were incubated with Dylight 488 conjugated Goat Anti-Rabbit IgG (H + L) secondary antibody or Dylight 594 conjugated Goat Anti-Mouse IgG (H + L) secondary antibody in the dark for 2 h. Next, DNA was stained with DAPI (4′,6-diamidino-2-phenylindole, Beyotime Biotechnology, China) for 5 min. Finally, the cell slides were sealed with Antifade Mounting Medium and stored in the dark at 4°C. Immunofluorescence images were obtained using a fluorescence microscope.
Co-Immunoprecipitation
MG63 cells were co-transfected with SIRT2 (penter-SIRT2-C-Flag-His) and Snail (pcDNA3.1-Snai1-c-HA) plasmids. Total proteins were extracted by weak RIPA lysis buffer (Beyotime, China). ProteinA/G Plus Magnetic Beads (MedChemExpress, USA) was pre-incubated with the IgG (Normal Rabbit IgG, B900610, Proteintech), SIRT2 or Snail antibody for 60 min on a spinning wheel at 4°C. The bead-antibody complexes were washed 3 times and suspended with the protein lysate on a spinning wheel at 4°C overnight. The beads were washed 4 times with PBST buffer, and were collected by magnetic Stand. The immunoprecipitates were then eluted by boiling with 2×loading buffer for Western blot analysis.
Xenograft tumorigenesis and metastasis
Six-week-old male BALB/c athymic nude mice were subcutaneously injected into the flanks with 1×107 MG63-shSIRT2 cells or MG63-shNC cells in 0.1 mL of PBS (n = 6 per group). Tumor growth was monitored and volume was measured every 2 days with calipers. The tumor volume was calculated with the formula: (length×width 2)/2. Twelve days after injection, the mice were sacrificed, and tumors were harvested and weighed, then fixed in formalin and embedded in paraffin. The H&E staining and immunochemical staining of SIRT2 and Snail were performed. For tumor metastatic assay, 2×106/100uL MG63-shSIRT2 cells or MG63-shNC cells were injected into the tail vein of nude mice (n = 6 per group). Five weeks later, experimental lung and liver metastasis was determined. All animal experimental procedures and protocols were approved by the Experimental Animal Ethics Committee of Shandong University School of Medicine.
Statistical analysis
Data were expressed as the mean ± standard deviation (SD). Statistical analysis was carried out using SPSS 20.0 (IBM, Chicago, IL, USA). The significant differences between the two groups were assessed by two-tailed Student’s t test. Values of P < 0.05 were considered as statistical significance.