Cell culture and reagents
Two human osteosarcoma cell lines (MNNG-HOS,143B) and human osteoblast hFOB1.19 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The human osteosarcoma U-2OS cell line were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured under standard conditions. Human osteosarcoma cells were cultured at 37℃ in a 5% CO2 atmosphere. Meanwhile, the standard culture conditions were used for hFOB1.19 cells (34.5℃ in a 5% CO2 atmosphere). Antibodies against ICT1 (AP20382b; Abgent, San Diego, CA, USA), Cleaved Caspase-3 (Immunoway, YT6161), Cleaved Caspase-9 (Immunoway, YP0598), BCL-2 (ab32124; Abcam, Cambridge, UK) and BAX (ab32503; Abcam, Cambridge, UK) were used.
Establishment of stable ICT1 knockdown cell lines and BCL-2 oversxpression cell lines
The OBiO Technology (Shanghai, China) provided plasmids containing sh-ICT1 or BCL-2 and a negative control plasmid. The shRNA sequences for ICT1 are 5'-GCTGTTAATGCTTGTCTATAACTCGAGTTATAGACAAGCATTAACAGC-3' and 5'-GCAGAATGTGAACAAAGTGAACTCGAGTTCACTTTGTTCACATTCTG C-3'. The overexpression RNA sequence for BCL-2 is : 5′-GTTGTAGCGGGACAC CTACTGAAAGTTCTCTTCAGTAGGTGTCCCGCTACAAAAAAACTTA-3′.HEK 293T cells were used to package these plasmids into virus particles and the viral titers were measured. In order to establish stable ICT1-knockdown cells and BCL-2 overexpression cells, the target cells were infected with 1 × 108 lentivirus-transducing units with 6 mg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). Then,infected cells were screened with 2.5 mg/mL puromycin after 72 h. The efficiency was determined by western blotting and qRT-PCR.
RNA Isolation And qRT-PCR
Total RNAs were extracted and reverse transcription were performed as described before[18]. ICT1, BAX and BCL-2 transcripts were quantified with the SYBR® Premix Ex Taq™ kit (Takara Bio, Otsu, Japan). The primers used for detecting the messenger RNA (mRNA) expression levels of BCL2, BAX and ICT1 are as follows: BCL-2: forward 5′-GCCCTGTGGATGACTGAGTA-3′, reverse5′-TTCAGAGACA GCCAGGAGAAA-3′; BAX: forward 5′-GCTGGACATTGGACTTCCTC-3′, reverse 5′-GGCGTCCCAAAGTAGGAGAG-3′; ICT1: forward5′-CAGCCTGGACAAGCTC TACC-3′, reverse: 5′-GGAACCTGACTTCTGCCTTG-3′. The reaction conditions were: 95 ℃ for 5 min, 95 ℃ for 5 sec, 60 ℃ for 30 sec, and the fluorescence signal was detected from 40 cycles. β-actin and 18S were used as internal controls.
Western Blotting
Total cellular protein was extracted by using a protein extraction buffer (Beyotime, Shanghai, China). Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose (NC) membrane. After blocking, the membranes were probed with the following specific antibodies: anti-ICT1 (1:1000), anti-BCL-2 (1:1000), anti-BAX (1:1000), Cleaved Caspase-3 (1:10000), Cleaved Caspase-9 (1:1000) and anti-β-actin (1:2000), followed by species-specific secondary antibodies (1:10000). Finally, the bands were detected using a western electrochemiluminescence substrate (Share-bio, Shanghai, China).
Immunohistochemical Staining
A microarray containing tissue from 40 OS patients was obtained from Alena Biotechnology Co., Ltd. (Xi’an, China). The IHC assay was carried out as previously described[19]. Ki67 were detected employing the corresponding primary antibodies at 1:200 dilutions. We took photos of all the sections using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Scoring was measured according to the ratio of positive staining cells: 0–5% scored 0; 6–35% scored 1; 36–70% scored 2; more than 70% scored 3 and staining intensity: no staining scored 0, weakly staining scored 1, moderately staining scored 2 and strongly staining scored 3. The final score was determined using the percent of positive cell score × staining intensity score as follows: a score of 0–1 is “–”, a score of 2–3 is “+”, a score of 4–6 is “+ +” and a score of > 6 is “+ + +”; a total score < 4 was defined as low expression and a total score ≥ 4 was defined as high expression. These scores were judged independently by two senior pathologists in a blinded manner.
Cell Counting Kit-8 (CCK-8) Assay, Colony Formation Assay
The CCK-8 assay was carried out following the vendor’s instructions (Dojindo Molecular Technologies, Japan). Briefly, cells (3 × 103) were seeded into 96-well plates. the absorbance at a wavelength of 450 nm was measured using a plate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 0, 24, 48, 72, 96, and 120 h.
The infected cells were seeded into a 6-well plate at an initial cell density of 1 × 103 cells/well. Colonies were washed with ice-cold PBS after two weeks. Then, the cell pellet was fixed with paraformaldehyde and stained with 0.5% (w/v) crystal violet. Finally, we took photographs and counted cell numbers.
Cell Apoptosis
For cell apoptosis analysis, the adherent cells were separated with 0.25% trypsin (without ethylenediaminetetraacetic acid) after culturing for 24 h in serum-free medium. Cells were stained with the Annexin V/FITC Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s explanations and analyzed by flow cytometry.
Xenograft Tumor In Nude Mice
The BALB/C nude mice (no sex limitation; 5-week-old) were fed and all animal experiments were taken place in the East China Normal University and were carried out according to the animal experimental protocols approved by the East China Normal University Animal Care Commission. The mice were randomly assigned into several groups. In brief, the BALB/C nude mice were anaesthetised with 1.5% pentobarbital sodium. Then, U-2OS cells (1.5 × 106) were subcutaneously inoculated into mice. Tumor size was measured every 5 days. After 20 days, all mice were euthanized by CO2. Tumors were removed, the weight and size of which were recorded.
Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick-end Labeling (TUNEL) Assay
The proportion of apoptotic cells in the xenograft tumors was quantified using the TUNEL kit (Roche, Basel, Switzerland). This assay was performed as previously described[20].
Statistical Analyses
The Prism 5.0 software for Windows (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses. The expression of data was represented as mean ± SD. Student's t-test was used to perform the comparison between different groups. A p-value < 0.05 was considered statistically significant.