The glioma cell line U251 and U87 MG cells were obtained from Type Culture Collection of Chinese Academy of Sciences and American Type Culture Collection, respectively. The U251 cells were maintained in DMEM (Gibco, Carlsbad, CA, USA). The U87 MG cells were cultured in MEM (Gibco). All the cells were supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 µg/ml streptomycin and 100 U/ml penicillin (Hyclone), cultured at 37°C, in a humidified environment of 5% CO₂ atmosphere.

The sequences of SPOCK1 shRNA and negative control (NC) shRNA were 5′-GUAAUGAGGAGGGCUAUUA-3′ and 5′-TTCTCCGAACGTGTCACGT-3′, respectively. The sh-SPOCK1 and sh-NC were cloned into pGCsi-H1 vector (Genechem, Shanghai, China) separately and transfected into U87 MG cells. The human full-length cDNA of SPOCK1 was inserted into pEGFP-N1 vector (Clontech, San Jose, CA, USA) and transfected into U251 cells. Control cells were transfected with pEGFP-N1 vector. After transfection for 24 h, 400 µg/ml G418 was added into U87 MG and U251 cells for 2 weeks to select stable SPOCK1-silencing/overexpressing clones. The mRNA and protein expression levels of SPOCK1 were determined by qPCR and western blotting.

Rabbit anti-cleaved caspase-3 (1:1000), anti-cleaved PARP (1:1000) antibodies were purchased from Abcam (MA, USA). Rabbit anti-Bax (1:400), anti-Bcl-2 (1:400), anti-PI3K (1:400), anti-β-catenin (1:400), anti-MMP2 (1:400), anti-MMP9 (1:400), mouse anti-c-MYC (1:200), anti-cyclin D1 (1:400) were obtained from Boster (Wuhan, China). Mouse anti-SPOCK1 (1:200), rabbit anti-p-AKT (1:200), anti-AKT (1:200), anti-Wnt (1:200) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-p-PI3K (1:500) were obtained from Bioss (Beijing, China). Briefly, the cells were lysed in RIPA and then denatured. The protein concentration was measured using the BCA protein estimation kit (Beyotime, Shanghai, China). Protein samples were separated on an SDS-polyacrylamide gel and transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in PBS for 2 h at room temperature, and incubated with primary antibodies, respectively, at 4°C overnight. After incubation with a secondary antibody the blots were visualized by ECL detection reagent (Beyotime).

Quantitative PCR was used to quantify the expression of mRNA in cultured cells. Briefly, total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The total RNA (1 µg) was reverse transcribed by a reverse transcriptase (Takara, Shiga, Japan). RNA expression was measured by qRT-PCR using the SYBR-Green method (Takara) according to the manufacturer's instructions. The primer sequences of SPOCK1 were as follows: SPOCK1 (forward, 5′-CACTGGGTTGGACCTTCGA-3′; reverse, 5′-CTTTGGTGGCTCAGGCTCT-3′) and β-actin (forward, 5′-CTTAGTTGCGTTACACCCTTTCTTG-3′; reverse, 5′-CTGTCACCTTCACCGTTCCAGTTT-3′). β-actin was used as an internal reference gene to normalize the expression of detection genes. Relative quantification of gene was analyzed by the comparative threshold cycle (Ct) method.

Cell proliferation was determined by Cell Counting Kit-8 (CCK8) (Beyotime, Shanghai, China). Cells were seeded into 96-well plates (3×10³ per well) and incubated for 24, 48, 72, 96 h, respectively. After incubation, 10 µl of CCK8 reagent was added to each well and at 450 nm the absorbance was detected by a microplate reader (Bio-Tek, Winooski, VT, USA). The results represent three independent experiments.

For the cell cycle assay, the cells in exponential growth period were collected, washed twice with cold PBS, fixed in cold 70% ethanol at −20°C. After washing with cold PBS the cells were incubated with 10 mg/ml Rnase A (Beyotime) and 1 mg/ml propidium iodide (Beyotime) at 37°C for 30 min. Cell cycle was performed by flow cytometry (BD Biosciences, San Jose, CA, USA).

Cells were seeded into a six-well plate (200 cells per well). Cells were cultured at 37°C with 5% CO₂ for 14 days until the clones were visible to the naked eye. Thereafter, the medium was removed and the cells were fixed with 4% paraformaldehyde, dyed with crystal violet. Under a microscope (Olympus, Tokyo, Japan) stained clones with cell number >50 were counted and digital images were taken.

Cells grown on cover slips were fixed with 4% paraformaldehyde for 15 min at room temperature. Then the cells were permeabilized with 0.1% Triton-X-100 solution for 30 min. After washing with PBS, cells were blocked with 10% goat serum for 15 min at room temperature. Then, cells were incubated with mouse monoclonal anti-PCNA (1:50) in PBS with 10% goat serum at 4°C overnight. After washing with PBS three times, cells were incubated with Cy3-conjugated anti-mouse secondary antibodies for 1 h at room temperature. The nuclei were counterstained with DAPI (Biosharp, Seattle, WA, USA). Under a magnification of ×400, fluorescence images of 5 different microscopic fields were captured by a fluorescence microscope.

Cells were seeded in 12-well plates and incubated until >80% confluence. A straight wound was created by scratching with a 200-µl pipette tip. Floating cells were removed by washing with serum-free medium twice. The cells were then cultured in serum-free medium and allowed to migrate into the wound area. Images of the migrated cells were acquired with an inverted microscope (Olympus) at 0, 12, 24 h.

Transwell chambers (Corning Inc., Corning, NY,. USA) were pre-coated with matrigel (BD Biosciences) at 37°C for 2 h. Cells (10⁴) in 200 µl serum-free medium were added to the upper compartment, and to the lower chamber was added 800 µl DMEM containing 20% FBS. Then the cells were incubated for 24 h at 37°C with 5% CO₂. A cotton swab was used to remove the non-invaded cells in the upper compartment. The invaded cells were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. Under the microscope the cells were counted in five random sights.

The samples were separated in 10% SDS polyacrylamide gel containing 0.2% gelatin in ice bath for 2.5 h. Then the gels were washed in eluent buffer (2.5% Triton X-100, 50 mM Tris-HCl, 5 mM CaCl₂, 1 µM ZnCl₂, pH 7.6) for 40 min, twice; equilibrated in developing buffer (50 mM Tris-HCl, 5 mM CaCl₂, 1 µM ZnCl₂, pH 7.6) for 20 min, twice; and finally put in incubation buffer (50 mM Tris-HCl, 5 mM CaCl₂, 1 µM ZnCl₂, 0.02% Brij, 0.2 M NaCl) at 37°C for 40 h. Then, the gels were incubated with staining buffer (0.05% Coomassie blue G-250 in 45% methanol, 10% acetic acid, 30% methanoic acid) for 3 h and then washed with destaining buffer (45% methanol, 10% acetic acid) until clear bands appeared. The images were obtained by gel imaging system (Bio-Rad, Hercules, CA, USA) and the activities of MMPs were measured by densitometric analysis.

Annexin V-FITC/PI staining was used to measure alive, apoptotic and necrotic cells. Briefly, cells were harvested, washed with PBS and stained with a mixture of 100 µl Annexin V-FITC and PI in the dark for 15 min. Then flow cytometry was used to classify fluorescent cells. The number of apoptotic cells was analysed by BD FACSuite software.

Hoechst 33342 staining was performed to measure apoptotic morphology. Briefly, 5×10⁴ cells were seeded on cover slips and fixed with 4% paraformaldehyde for 20 min at room temperature. After washing twice with PBS, the cells were stained with Hoechst 33342 for 5 min. Fluorescent images were acquired by a fluorescence microscopy (Olympus).

All results are expressed as mean ± standard deviation (SD). Statistical analyses were performed using Student's t-test. A p-value of <0.05 was set as the significance level.

To investigate the role of SPOCK1 in glioma cells, we chose U87 MG cells with higher SPOCK1 expression level for stable transfection with shRNA vector toward SPOCK1 and U251 cells, in which SPOCK1 is infrequently expressed, for stable transfection with SPOCK1 expression vector. The expression levels of SPOCK1 was measured by real-time PCR and western blot analysis. As shown in Fig. 1A and C, an efficient silencing of SPOCK1 protein and mRNA expression was shown in U87 MG cells transfected with the SPOCK1 shRNA compared with negative control group. An obvious high level of SPOCK1 protein and mRNA expression was apparent in U251 cells transfected with SPOCK1 expression vector (Fig. 1B and D).

The effect of SPOCK1 on the proliferation of glioma cells were measured by CCK8 assay. As shown in Fig. 2A, silencing of SPOCK1 significantly inhibited the proliferation of U87 MG cells. While overexpression of SPOCK1 could promote the proliferation of U251 cells. Additionally, the ability of glioma cells to form colonies was also determined. As shown in Fig. 2B, the result showed that silencing of SPOCK1 significantly decreased the number of colonies formed by U87 MG cells. Overexpression of SPOCK1, on the contrary, increasing colony formation in U251 cells. Moreover, the cell cycle progression was detected by flow cytometric analysis (Fig. 2C). The results demonstrated that silencing of SPOCK1 resulted in a larger fraction of the population in the G1 phase and a significant decrease in the proportion in G2/M phase. Overexpression of SPOCK1 significantly reduced the number of cells arrested in G1 phase. Since PCNA is an index for evaluating the ability of cell proliferation, we subsequently detected the expression of PCNA by immunofluorescence assay. As shown in Fig. 3, knockdown of SPOCK1 significantly downregulated the expression of PCNA in U87 MG cells. On the contrary, overexpression of SPOCK1 upregulated the expression of PCNA in U251 cells. The above findings provide evidence that SPOCK1 as an oncogene may promote glioma cell growth.

To further explore the mechanism of SPOCK1 in tumor growth, we focused on the role of SPOCK1 in cell apoptosis. As assessed by Annexin V-FITC and PI staining and shown in Fig. 4A, knockdown of SPOCK1 induced obvious apoptosis in U87 MG cells. While the apoptosis was inhibited by SPOCK1 overexpression in U251 cells. Moreover, as shown in Fig. 4B, the nuclear morphological changes in the apoptotic cells were revealed by the Hoechst 33342 staining. Silencing of SPOCK1 resulted in brighter chromatin condensation and nuclear fragmentation of the nuclei, which was significantly suppressed by SPOCK1 overexpression. To further confirm the effect of SPOCK1 on apoptosis, a number of apoptosis related proteins were determined. The results showed that knockdown of SPOCK1 induced increase in expression levels of Bax, cleaved PARP and cleaved caspase-3 and decreased expression of Bcl-2 in U87 MG cells. The expression changes of these apoptosis related proteins were inverted when SPOCK1 was overexpressed in U251 cells. These data indicated that SPOCK1 promotes glioma cell growth by reducing cell apoptosis.

Cell migration capacity was assessed by wound healing assay. As shown in Fig. 5A, knockdown of SPOCK1 obviously inhibited the migration capacity of U87 MG cells. When SPOCK1 was overexpressed in U251 cells, the migration capacity was increased significantly. Moreover, the invasive capacity was assessed by transwell assay and shown in Fig. 5B. The results were similar to the changes in migration ability that was inhibited by SPOCK1 silencing and promoted by SPOCK1 overexpression. To further confirm the effect of SPOCK1 on metastasis, the activity and expression of MMP9 and MMP2 was determined by gelatin zymography and western blotting. As shown in Fig. 6A, the activity of MMP9 and MMP2 was restrained by SPOCK1 silencing in U87 MG cells. While overexpression of SPOCK1 could promote the activity of MMP9 and MMP2. The changes of MMP9 and MMP2 expression were similar to those of MMP9 and MMP2 activity (Fig. 6B).

Since PI3K/AKT and Wnt/β-catenin signaling pathways are associated intimately with proliferation and metastasis of cancer cells, the effect of SPOCK1 on PI3K/AKT and Wnt/β-catenin signaling pathways was evaluated. As shown in Fig. 7, knockdown of SPOCK1 significantly suppressed the protein levels of p-PI3K, p-AKT, Wnt and β-catenin in U87 MG cells, which could be reversed when SPOCK1 was overexpressed in U251 cells. Moreover, the downstream targets of Wnt/β-catenin were also assessed. The results indicated that the protein levels of c-MYC and cyclin D1 were decreased by SPOCK1 silencing, which were increased obviously when SPOCK1 was overexpressed.