Thirty-five formalin-fixed, paraffin-embedded osteosarcoma specimens (before the administration of neo-adjuvant chemotherapy) were collected according to Chinese National Ethical Guidelines (‘Code for Proper Secondary Use of Human Tissue’, Chinese Federation of Medical Scientific Societies). All of the patients exhibited the diagnostic criteria for osteosarcoma as defined by the World Health Organization classification. Written informed consent was obtained from each patient before entering the present study, and all study protocols were approved by the Ethics Committee for Clinical Research of Wuhan University (Wuhan, China). The clinical data of IDH-1 were described in our previous study (22). Osteosarcoma specimens were graded using Rosen classification (23) (grade 1, tumor necrosis <50%; grade 2, tumor necrosis 50–90%; grade 3, tumor necrosis >90%; grade 4, tumor necrosis 100%; grade 1 and 2 are low histological Rosen grades, and grade 3 and 4 are high histological Rosen grades). We obtained 4 cases of grade 1, 12 cases of grade 2, 13 cases of grade 3, and 6 cases of grade 4. All of the osteosarcoma specimens were sliced into 4-µm serial sections followed by immunohistochemical staining.

All of the sections were obtained from formalin-fixed, paraffin-embedded osteosarcoma tissues and baked at 65°C for 2 h. After dewaxing, the sections were washed with phosphate-buffered saline (PBS) 3 times, and then placed in EDTA buffer for microwave antigen retrieval, boiled at medium heat and 10 min later, boiled again at low heat. After cooling, the sections were washed again with PBS 3 times, and then placed in 3% hydrogen peroxide solution and incubated for 10 min at room temperature in the dark. Anti-IDH-1 rabbit polyclonal antibody (Protein Tech Group, Chicago, IL, USA) or anti-HIF-1α rabbit polyclonal antibody (Abcam, Cambridge, MA, USA) was added to each section to cover the tissue and kept at 4°C overnight. Then, a goat anti-rabbit secondary antibody (KPL, Gaithersburg, MD, USA) was added to each section and incubated at 37°C for 50 min. The sections were washed with PBS 3 times for 5 min each time. Following the removal of PBS, 50–100 µl fresh DAB solution (Zymed, South San Francisco, CA, USA) was added to each section. Chromogenic reactions were controlled with a microscope, and hematoxylin staining was performed. A brown or tan zone was considered positive staining. The percentage of positive staining cells in the specimens was used to score the immunohistochemistry staining results (24,25) (for each section, two double-blinded pathologists independently observed each slide, 10 fields were randomly selected, and the mean positive cell percentage was used as the final result), and the specific classification was as follows: 0, no positively stained cells; 1 point, a positive cell percentage (i.e., cell staining rate) ≤10%; 2 points, a positive cell percentage of 10–25%; 3 points, a positive cell percentage of 26–50%; 4 points, a positive cell percentage of 51–75%; and 5 points, a positive cell percentage of >75%. A staining rate of 10% was chosen as the critical value of the negative and positive protein expression, and a staining rate >10% was considered positive protein expression and ≤10% was negative. Zero to 3 points denoted a low expression level (cell staining rate ≤50%), and 4–5 points represents a high protein expression level (cell staining rate >50%).

Human osteosarcoma cell lines MG-63 and 143B were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) through LGC Promochem (Wesel, Germany). Osteosarcoma cells were exposed to hypoxia (oxygen content of 1%) and normal conditions (oxygen content of 20%) (26). The cells were thoroughly lysed with an appropriate amount of RIPA lysis buffer (Aspen, USA), and the lysate was centrifuged at 4°C at 12,000 rpm for 5 min to collect the total protein solution. Using a BCA protein assay kit (Aspen), the protein concentration of the sample was determined. According to the concentration of each sample, 40 µg of total protein of each sample was used as a loading sample, in which the appropriate amount of 5X protein loading buffer was added, and then heated in a 95°C water bath for 5 min and loaded on a gel. After the electrophoresis was complete, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The transferred membranes were blocked with 5% skim milk in Tris-buffered saline with 0.05% Tween-20 (TBST) and washed 6 times in TBST. IDH-1 and HIF-1α proteins were detected by the rabbit polyclonal antibody for IDH-1 (Protein Tech Group) or HIF-1α (Abcam). β-actin proteins were recognized by the β-actin-specific monoclonal mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Antibodies were diluted according to the manufacturers instructions, and were incubated overnight at 4°C followed by incubation with peroxidase-conjugated goat anti-rabbit immunoglobulin (1:2,000; Santa Cruz Biotechnology) in TBST for 1 h. Signals were developed using enhanced chemiluminescent reagent (Pierce Biotechnology, Rockford, IL, USA). β-actin was used as the internal loading control. The band intensity was analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA). Relative expression was calculated as the intensity ratio of target protein to that of β-actin.

To downregulate HIF-1α, a small interfering RNA sequence inserted into the pLL3.7 shRNA lentivector (Genesil, Wuhan, China) to reduce HIF-1α expression was 5′-CUGAUGACCAGCAACUUGAdTdT-3′ (sense) and 5′-UCAAGUUGCUGGUCAUCAGdTdT-3′ (antisense) (27). The empty lentivector (Genesil) was used as a negative control. Non-lentivector infection was used as an additional control. The lentivector stocks were added to the osteosarcoma cell line MG-63. The cells were infected with the lentivector, and the GFP-positive cells were counted before G418 selection, using 800 µg/ml for MG-63. The efficiency of the highest infection, as determined by directly counting GFP-positive cells and G418 selection, was obtained at a multiplicity of infection (MOI) of 50 for MG-63. The frequency of infection rose to 95% for MG-63 cells. Cells infected with knockdown lentivector (KD) were called KD cells. Cells without lentivector infection were called NT cells, and the cells with empty lentivector infection were called NC cells. After selection, the efficiency of infection was verified by western blotting.

A total of 3.5×10³ cells were seeded in each test well into a 96-well plate to detect cell growth. The cells were washed with PBS after cultured for 1–6 days. MTT (5 mg/ml) was then added to each well, and the mixture was incubated at 37°C for 4 h. The culture medium was then replaced with an equal volume of dimethyl sulfoxide (DMSO). After shaking the plate at room temperature for 10 min, the absorbance of each well was determined at 570 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

All of the data were analyzed using SPSS 20.0 statistical software. Numerical variable data with homogeneity of variance were analyzed by an independent samples t-test, and non-normal numerical variable data were analyzed using the Mann-Whitney U test. Categorical variables were analyzed using a Chi-square or Fisher's exact probability tests while a correlation test of normal distribution data was analyzed by the Pearson correlation coefficient method, and that of the non-normal data was performed using the Spearman correlation coefficient method. The results with P<0.05 were considered to be statistically significant and those with P<0.01 were of high statistical significance.

There were no significant differences in HIF-1α expression levels between groups in regards to sex, age, primary tumor location and tumor tissue type (P>0.05) (Table I). The IDH-1 expression level in the low histological Rosen grade group (tumor necrosis, ≤90%) was significantly higher than that in the high histological Rosen grade group with a statistically significant difference (P<0.01), and the expression level and the tissue classification was negatively correlated (r=−0.427) (Fig. 1). In contrast, HIF-1α expression in the high histological Rosen grade group was significantly higher than that in the low histological Rosen grade group, and was positively correlated with the tissue classification (P<0.05, r=0.356) (Fig. 1). Our results also showed that the survival rate was not significantly correlated with the expression of HIF-1α (P>0.05) (Fig. 2).

We already described that IDH-1 is mainly located in the cytoplasm (22). In the present study, we found that HIF-1 protein was also significantly expressed in the cytoplasm (Fig. 3). The HIF-1α protein staining rate ranged from 5 to 80%, with a mean value of 41.46% and an SD of 24.81%, and the corresponding positive staining score was 1–5, with a mean value of 3.00 and an SD of 1.33. The positive expression rate of HIF-1α protein was 77.1% (27/35) with a high expression rate of 40% (14/35). The correlation test between the IDH-1 and the HIF-1α expression levels in 35 samples revealed P<0.01 and r=−0.700 (Fig. 4), indicating that the IDH-1 protein expression level was negatively correlated with that of the HIF-1α protein.

Our results showed that the expression of IDH-1 protein in 143B and MG-63 cells under normal conditions was higher than that under hypoxia, and the differences were statistically significant (P<0.05) (Fig. 5A and C). In contrast, HIF-1α proteins in 143B and MG-63 cells under hypoxic conditions were both highly expressed compared to that in normal conditions (P<0.05) (Fig. 5B and C).

HIF-1α downregulation decreased the cell growth rate of MG-63 KD cells by 0.6- and 0.5-fold on day 6 compared to the MG-63 NC cells under normal conditions and hypoxic conditions, respectively (P<0.01) (Fig. 6). There was no significant difference in the cell growth rate between MG-63 NT and MG-63 NC cells either under normal conditions or hypoxic conditions. The MG-63 cell growth rate was suppressed under hypoxic conditions.

Lentivectors were used to downregulate HIF-1α and the infection efficiency was confirmed by western blotting. HIF-1α was significantly decreased in the MG-63 KD cells, compared with the NT and NC cells (P<0.01) both in the hypoxic and normal group (Fig. 7B and C). There was no significant difference in HIF-1α protein expression between NT and NC cells (P>0.05). Meanwhile, IDH-1 protein expression in the MG-63 KD cells was significantly higher than that noted in the NT and NC cells either under normal or hypoxic conditions (P<0.01) (Fig. 7A and C).