The human OS (MG-63 and U-2 OS) cell lines used in the experiments were from the Institute of Biochemistry and Cell Biology (Shanghai, China). Lentiviral-mediated GAL1 shRNA (Lv-shGAL1) vector, negative control vector and virion-packaging elements were from GeneChem (Shanghai, China). The primer of GAL1 was synthesized by ABI (Framingham, MA, USA). All antibodies used were purchased from Cell Signaling Technologies (Boston, MA, USA).

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Thermo Fisher Scientific Inc. (Waltham, MA, USA); TRIzol reagent and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA, USA); M-MLV reverse transcriptase was from Promega (Madison, WI, USA); SYBR-Green Master Mixture was from Takara (Otsu, Japan). Cell apoptosis kit [propidium iodide (PI), RNase A, Annexin V-FITC] was from KeyGen Biology (Nanjing, China). The ECL-Plus kit was from GE Healthcare (Piscataway, NJ, USA).

Human OS tissues and the corresponding adjacent non-cancerous tissues (ANCT) were obtained from 30 consecutive cases admitted to our hospital from January 2006 to December 2010. The present study was approved by the Medical Ethics Committee of Shanghai University of Traditional Chinese Medicine, and written informed consent was obtained from the patients or their parents before sample collection. Two pathologists respectively reviewed all of the cases.

GAL1 antibody was used for IHC detection of protein expression in the tissue microarrays. GAL1 antibody was used at a 1:100 dilution. Endogenous peroxidase was inhibited by incubation with freshly prepared 3% hydrogen peroxide with 0.1% sodium azide. Non-specific staining was blocked with 0.5% casein and 5% normal serum. Tissue microarrays were incubated with biotinylated antibodies and horseradish peroxidase. Staining was developed with diaminobenzidine substrate and sections were counterstained with hematoxylin. Phosphate-buffered saline (PBS) replaced the GAL1 antibody in the negative controls. The expression of GAL1 was semi-quantitatively estimated as the total immunostaining scores. Expression of GAL1 in each specimen was scored according to the percentage of positively stained cells counted in five randomly selected high magnification fields: (−) no expression; (+) positive cell ratio ≤25%; (++) positive cell ratio 26–50%; and (+++) positive cell ratio >50%.

MG-63 and U-2 OS cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml of penicillin and 100 μg/ml of streptomycin. They were all placed in a humidified atmosphere containing 5% CO₂ at 37°C. Lv-shGAL1 and the negative control virus were transfected into OS cells. Cells were subcultured at a 1:5 dilution in 300 μg/ml G418-containing medium. Positive stable transfectants were selected and expanded for further study. The clone in which the Lv-shGAL1 vector was transfected was named the Lv-shGAL1 group, and the negative control vector transfected clone was named the NC group.

To quantitatively determine the mRNA expression level of GAL1 in OS cell lines, real-time PCR was used. Total RNA of each clone was extracted with TRIzol according to the manufacturer’s protocol. Reverse-transcription was carried out using M-MLV, and cDNA amplification was carried out using the SYBR-Green Master Mix kit according to the manufacturer’s protocol. The GAL1 gene was amplified using specific oligonucleotide primers, and the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control. The PCR primer sequences were as follows: GAL1, 5′-GCGTGGCTG CTGGGAGGTATC-3′ and 5′-GGAACAGAAAGACTCCA ATG-3′; β-actin, 5′-CAACGAATTTGGCT-ACAGCA-3′ and 5′-AGGGGTCTACATGGCAACTG-3′. Data were analyzed using the comparative Ct method (2^−ΔΔCt). Three separate experiments were performed for each clone.

OS cells were harvested and extracted using lysis buffer (Tris-HCl, SDS, mercaptoethanol and glycerol). Cell extracts were boiled for 5 min in loading buffer, and then equal amounts of cell extracts were separated on 15% SDS-PAGE gels. Separated protein bands were transferred into polyvinylidene fluoride (PVDF) membranes and the membranes were blocked in 5% skim milk powder. The primary antibodies against p38MAPK, p-ERK, Ki-67, matrix metallopeptidase-9 (MMP-9) and caspase-3 were diluted according to the relevant instructions and incubated overnight at 4°C. Then, horseradish peroxidase-linked secondary antibodies were added at a dilution ratio of 1:1,000, and incubated at room temperature for 2 h. The membranes were washed with PBS for three times and the immunoreactive bands were visualized using the ECL-Plus kit according to the kit’s instructions. The relative protein level in the different groups was normalized to the GAPDH concentration. Three separate experiments were performed for each clone.

OS cells treated with Lv-shGAL1 were counted and seeded in 12-well plates (in triplicate) at 100 cells/well. Fresh culture medium was replaced every three days. Colonies were counted only if they contained >50 cells, and the number of colonies was counted from the 6th day after seeding. The cells were then stained using crystal violet. The rate of colony formation was calculated with the equation: Colony formation rate = (number of colonies/number of seeded cells) × 100%.

Transwell filters were coated with Matrigel (3.9 μg/μl, 60–80 μl) on the upper surface of a polycarbonic membrane (diameter 6.5 mm, pore size 8 μm). After incubation at 37°C for 30 min, the Matrigel solidified and served as the extracellular matrix for analysis of tumor cell invasion. Harvested cells (1×10⁵) in 100 μl of serum-free DMEM were added into the upper compartment of the chamber. Conditioned medium (200 μl) derived from NIH3T3 cells was used as a source of chemoattractant, and was placed in the bottom compartment of the chamber. After a 24-h incubation at 37°C with 5% CO₂, the medium was removed from the upper chamber. The non-invaded cells on the upper side of the chamber were scraped off with a cotton swab. The cells that had migrated from the Matrigel into the pores of the inserted filter were fixed with 100% methanol, stained with hematoxylin, and mounted and dried at 80°C for 30 min. The number of cells invading through the Matrigel was counted in three randomly selected visual fields from the central and peripheral portion of the filter using an inverted microscope (x200 magnification). Each assay was repeated three times.

To detect cell apoptosis, OS cells were trypsinized, washed with cold PBS and resuspended in binding buffer according to the instructions of the apoptosis kit. FITC-Annexin V and PI were added to the fixed cells for 20 min in darkness at room temperature. Then, Annexin V binding buffer was added to the mixture before the fluorescence was measured on a FACsort flow cytometer. Cell apoptosis was analyzed using Cell Quest software (Becton-Dickinson, USA). Three separate experiments were performed for each clone.

Six-week-old female immunodeficient nude mice (BALB/c-nu) were bred at the laboratory animal facility (Institute of Chinese Academy of Sciences, Shanghai), and were housed individually in microisolator ventilated cages with free access to water and food. Three mice were injected subcutaneously with 1×10⁷ OS cells (MG-63) in 50 μl of PBS pre-mixed with an equal volume of Matrigel matrix (Becton-Dickinson). Mice were monitored daily and developed subcutaneous tumors. When the tumor size reached ~5 mm in length, they were surgically removed, cut into 1–2 mm³ pieces, and re-seeded individually into other mice. When the tumor size reached ~5 mm in length, the mice were randomly assigned to the NC and Lv-shGAL1 group. In the treatment group, 15 μl of Lv-shGAL1 was injected into the subcutaneous tumors using a multi-site injection format. Injections were repeated every other day after initial treatment. The tumor volume every three days was measured with a caliper, using the formula: Volume = (length × width)²/2.

SPSS 20.0 was used for statistical analysis. Kruskal-Wallis H and Chi-square tests were used to analyze the expression rate in all groups. One-way analysis of variance (ANOVA) was used to analyze the differences between groups. The LSD method of multiple comparisons was used when the probability for ANOVA was statistically significant. Statistical significance was set at P<0.05.

Expression of GAL1 protein was evaluated using IHC staining. Positive expression of GAL1 protein was examined in the nucleus and cytoplasm of OS tissues and ANCT (Fig. 1), and positive GAL1 expression was detected in 63.3% (19/30) of OS tissues, compared with a positive rate of 36.7% (11/30) in ANCT (P=0.029) (Table I).

The association between GAL1 expression and various clinicopathological factors was analyzed. As shown in Table II, increased expression of GAL1 was closely correlated with distant metastasis of OS (P=0.022). However, no significant association was found between GAL1 expression and other factors including age, gender of the patients, and histology and Ennecking staging of the tumor (P>0.05, respectively).

Real-time PCR showed a significantly lower level of GAL1 mRNA in the Lv-shGAL1 group than that in the NC group (each P<0.01) (Fig. 2A and B). The protein expression levels of GAL1, p-MAPK and p-ERK, as indicated by western blot assay were markedly decreased in the Lv-shGAL1 group in comparison with levels in the NC group (each P<0.01) (Fig. 2C–F).

In order to test the effect of GAL1 knockdown on cell growth, we investigated the independent growth of OS cells by colony formation assay. We found that knockdown of GAL1 significantly diminished the independent growth of OS cells (Fig. 3A–D, P<0.01). To determine whether knockdown of GAL1 suppressed the endogenous expression of Ki-67 through translational repression, the expression of Ki-67 protein was examined by western blotting, indicating that Ki-67 was decreased in the Lv-shGAL1 group compared with the expression level in the NC group (P<0.01) (Fig. 3E and F).

To determine the effect of GAL1 knockdown on cell invasion, a Transwell assay was carried out. The invasive potential was determined on the basis of the ability of cells to invade a matrix barrier containing laminin and type IV collagen, major components of the basement membrane. Representative micrographs of Transwell filters are shown in Fig. 4A and B. The invasive activity of OS cells was significantly reduced in the Lv-shGAL1 group when compared with that in the NC group (P<0.01) (Fig. 4C and D). The endogenous expression of MMP-9 protein, evaluated by western blotting, was significantly reduced in the Lv-shGAL1 group when compared to that in the NC group (P<0.01) (Fig. 4E and F).

To determine whether GAL1 knockdown affects cell apoptosis, flow cytometric analysis with PI/FITC-Annexin V staining was performed. The apoptotic rate of the OS cells was markedly higher in the Lv-shGAL1 group than that in the NC group (P<0.01) (Fig. 5A–D). The endogenous expression of caspase-3 protein, evaluated by western blotting, was significantly increased in the Lv-shGAL1 group when compared with the expression level in the NC group (P<0.01) (Fig. 5E and F).

Our in vitro experiments demonstrated the inhibitory effect of GAL1 knockdown on tumor growth. Thus, we further investigated the effect of GAL1 on MG-63 xenograft tumor growth in vivo. The mean volume of the tumors in the experimental mice before treatment was 75.85±19.35 mm³. During the entire tumor growth period, the tumor growth activity was assessed. The tumors treated with Lv-shGAL1 grew substantially slower when compared to the rate of growth in the NC group (Fig. 6A). When the tumors were harvested, the average weight and volume of the tumors were significantly smaller in the Lv-shGAL1 group than those of the NC group (Fig. 6B and C).