The human brain glioma cell lines LN229 and LN18 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), U87 and U251 were obtained from Sigma-Aldrich (St. Louis, MO, USA) and the normal human brain gliocyte cell line (HEB) was obtained from Shanghai, China. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin (all from Invitrogen, Carlsbad, CA, USA) and maintained at 37°C in a humidified incubator of 5% CO₂.

Total proteins were extracted using the Tissue or Cell Total Protein Extraction kit (Amresco, Solon, OH, USA) from LN229, LN18, U87 and U251 cell lines. All of the primary antibodies were purchased from Abcam (Cambridge, UK). The proteins were separated by SDS-PAGE followed by electrotransfer to an NC membrane; the membranes were probed using antibodies against SEPT7 (1:2,000), MMP-2 (1:1,000), MMP-9 (1:1,000), cofilin (1:1,000), phospho(ser3)-cofilin (1:1,000), and actin (1:5,000), followed by a horseradish peroxidase (HRP)-conjugated second antibody (GAPDH, 1:10,000) (Abcam). Bands were revealed with ECL reagent (Millipore, billerica, MA, USA) and recorded on X-ray film (Kodak, Xiamen, China). The densitometry of each band was quantified by a gel imaging system and Quantity One 4.62 software (Bio-Rad, Hercules, CA, USA).

Total RNA was extracted using TRIzol reagents (Invitrogen) from LN229, LN18, U87 and U251 cell lines. Isolated RNA was electrophoresed on 1% agarose gel to detect the purity of total RNA. The first-strand cDNA was synthesized using 1 µg total RNA and SuperScript^® III Reverse Transcriptase (Invitrogen). PCR amplification was performed using the PCR amplification kit (Takara Bio, Inc., Otsu, Japan). The specific primers were designed using Primer Premier 6.0 software and synthesized by Sangon biotech (Shanghai, China). The primers for SEPT7 were 5′-CTCTTGCTGTGGTAGGTAG-3′ (forward) and 5′-GCTTCTGTAGTTCTCATAGTG-3′ (reverse). The primers for GAPDH as an internal control were 5′-GAGTGAGTGGAAGACAGAAT-3′ (forward) and 5′-GCAGAGAAGCAGACAGTTA-3′ (reverse).

The pcDNA3.1(+)/SEPT7 expression vector was constructed by cloning SEPT7 fragment from normal human cDNA into pcDNA3.1(+) (Invitrogen) between BamHI and EcoRI sites to express SEPT7 in abundance in E. coli DH5α cells. The primers for SEPT7 were as follows: forward primer, 5′-GGAGGATCCCTCTTGCTGTGGTAGGTAG-3′ (including BamHI site GGATCC and three protection bases) and reverse primer, 5′-CTCGAATTCGCTTCTGTAGTTCTCATAGTG-3′ (including EcoRI site GGATCC and three protection bases). The recombinant plasmid was identified by endonuclease digestion and DNA sequencing. The pcDNA3.1(+)/SEPT7 and pcDNA3.1(+) plasmids were separately transfected into the glioma cells mediated by Lipofectamine 2000 (Invitrogen) as per the manual. The stably transfected clones were screened by G418, and identified by western blot analysis and RT-PCR. Finally, the siRNA or control siRNA was also transfected into the glioma cells mediated by Lipofectamine 2000 (Invitrogen) as manual. Within 24 h after transfection, the protein level was detected by western blot analysis and RT-PCR. The siRNA primers sequences were designed by Invitrogen Block-iT RNAi designer.

The cells were added into Transwell chambers (Millipore) and FBS was added into lower chamber. Cells were cultured at 37°C for 24 h in a humidified incubator of 5% CO₂, and then the hematoxylin and eosin (H&E) stained cells in the lower chamber were counted under the microscope. The cells in 10 high power fields were randomly chosen and the average transmembrane cell number per high-power field was shown as cell migration ability. For the cell chemotaxis assay, the different concentrations of IGF-1 (0, 1, 10 and 100 ng/ml) were added into the lower chamber of chemotaxis chambers (Millpore) with 30 µl per well. There were membranes with 8 µm gaps between the upper and lower chambers. Then, 50 µl of cell suspension with was added into the upper chamber of each well. After cells were cultured at 37°C for 24 h, the H&E stained cells in the lower chamber were counted under the microscope. The chemotactic index was presented as cell migration number under IGF-1 and without IGF-1.

The cells were broken, homogenized in cold lysis buffer (10 mM K₂HPO₄, 100 mM NaF, 50 mM KCl, 2 mM MgCl₂, 1 mM EgTA, 0.2 mM DTT, 0.5% Triton X-100, 1 mM sucrose, pH 7.0) and centrifuged at 15,000 × g for 30 min. Soluble actin (G-actin) was measured in the supernatant. The insoluble F-actin in the pellet was resuspended in lysis buffer plus an equal volume of buffer 2 (1.5 mM guanidine hydrochloride, 1 mM sodium acetate, 1 mM CaCl₂, 1 mM ATP and 20 mM Tris-HCl, pH 7.5) and incubated on ice for 1 h to convert F-actin into soluble G-actin, with gentle mixing every 15 min. The samples were centrifuged at 15,000 × g for 30 min, and F-actin was measured in this supernatant. Samples from the supernatant (G-actin) and pellet (F-actin) fractions were proportionally loaded and analyzed by western blot analysis using a specific actin antibody (Millipore, Temecula, CA, USA).

Data are presented as means ± SD. Statistical analysis was performed with SPSS13.0 software (IBM, Armonk, NY, USA). Statistical evaluation of the data was performed using one-way ANOVA and LSD for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

SEPT7 expression was identified by western blot analysis and RT-PCR in various glioma cells. The results showed that SEPT7 levels in glioma cells were reduced by 50% or less compared to normal human brain cells. SEPT7 levels in LN18 cell lines were lower than in other glioma cells (Fig. 1). LN18 was derived from a patient with a grade IV glioma in the right temporal lobe (ATCC). The subsequent tests therefore used the LN18 cell line as the research object.

To verify whether SEPT7 influences glioma invasion, a SEPT7 overexpression vector was constructed and transfected into the LN18 cell line (Fig. 2A and B). Analysis of SEPT7 expression in LN18 cells after transfection with SEPT7 overexpression plasmid indicated that the level of SEPT7 in pcDNA3.1/SEPT7 had a marked elevation compared to LN18 (P<0.01) (Fig. 2A and B). Malignant tumor invasion and metastasis mainly depend on cell migration, as well as extracellular matrix degradation and the induction of chemokines. Given the increased SEPT7 levels in pcDNA3.1/SEPT7, we examined whether a redundancy in SEPT7 alone accounts for the failure of glioma cells to invade and transfer. The results showed that the number of invading cells in the pcDNA3.1/SEPT7 group were significantly reduced in contrast to the LN18 group (P<0.01) (Fig. 2C), indicating that SEPT7 made no contribution to tumor cell migration. The effect of SEPT7 on tumor cell invasion and metastasis was further studied using an IGF-1-induced cell chemotaxis assay. Under the treatment of IGF-1 at different concentrations, glioma cell chemotaxis had a significant difference; overall, 10 ng/ml IGF-1 was the most powerful for glioma cell chemotaxis, either in LN18 or pcDNA3.1/SEPT7 (Fig. 2D). Consistent with cell migration results, the chemotaxis assay revealed that SEPT7 overexpression markedly diminished IGF-1 induced tumor cell chemotaxis compared to LN18 (P<0.01) (Fig. 2D). These results indicated that increased SEPT7 inhibits glioma cell migration and chemotaxis.

Activation of MMP-2 and MMP-9 regulates both matrix degradation and motility, thereby facilitating cellular invasion (16,17). The levels of MMP-2 and MMP-9 were probed with western blot analysis, indicating that the levels of MMP-2 and MMP-9 in pcDNA3.1/SEPT7 were markedly depressed, respectively, compared to LN18 (P<0.01 and P<0.05) (Fig. 2E). These results suggest that increased SEPT7 suppresses extracellular matrix degradation, consequently opposing cellular invasion.

SEPT7 is a cytoskeletal protein and is involved in the entry of axonal microtubules into nascent filopodia, enabling the formation of collateral branches (18). Increased SEPT7 binds to the actin filaments and promotes F-actin ring formation, against cell migration (19). Consistent with this conclusion, our study indicated that the F-actin/G-actin ratio in pcDNA3.1/SEPT7 was significantly reduced compared with the LN18 group (P<0.01) (Fig. 3A), revealing that SEPT7 upregulation can promote actin depolymerization.

Cofilin also acts as an actin-binding protein that plays an essential role in regulating actin filament dynamics and reorganization by stimulating the severance and depolymerization of actin filaments (14). Therefore, we speculated that a possible relationship exists between SEPT7 and cofilin phospho-regulation to modulate actin filament dynamics. To verify this conjecture, the levels of cofilin and p-cofilin were tested; the results showed that cofilin level in the pcDNA3.1/SEPT7 group decreased visibly compared with the LN18 group (P<0.01), while the p-cofilin level showed the opposite results (P<0.01) (Fig. 3B). These findings preliminary clarified that increased SEPT7 promotes the depolymerization of actin filaments, and is concerned with cofilin phospho-regulation.

To further explore the roles of cytoskeleton signaling in reduced SEPT7-mediated glioma cell migration, the control siRNA and SEPT7 siRNA were transfected into the LN18 cells. The degrees of transfection were tested at the protein and nucleic acid levels, revealing that the amount of SEPT7 was markedly reduced compared with the LN18 group (P<0.01 and P<0.01) (Fig. 4A and B). Cofilin, F-actin/G-actin ratio and cell migration in the SEPT7 siRNA group were significantly improved compared with the LN18 group (P<0.05, P<0.01 and P<0.01) (Fig. 4C–E), while p-cofilin expression reduced (P<0.01) (Fig. 4C). Thus, these experiments show that the knockdown of SEPT7 enhances the motility of glioma cells through cofilin phospho-regulation, which accelerates actin polymerization.

In view of the critical roles of SEPT7 and cofilin in linking signaling pathways to actin cytoskeleton remodeling, we further tested whether cofilin is involved in the process of SEPT7 siRNA-induced actin aggregation. The different cofilin siRNA were designed and transfected, respectively, into the LN18 cell lines, and the degrees of transfection were tested, showing that cofilin siRNA_579 was the best for inhibiting cofilin protein expression (P<0.01) (Fig. 5A). Afterwards, the cofilin siRNA_579 was transfected, respectively, into the normal, LN18, control siRNA and SEPT7 siRNA group cells. The cell migration and cytoskeleton locomotion were measured; as shown in Fig. 5B, the levels of cofilin and p-cofilin after cofilin inhibition were reduced in general in all groups compared with unrestrained cofilin (Figs. 4C and 5B). However, the level of cofilin in the SEPT7 siRNA group was further reduced compared with the LN18 group (P<0.01), while the p-cofilin level was just the opposite (P<0.01) (Fig. 5B).

Importantly, the F-actin/G-actin ratio and cell migration under cofilin inhibition in SEPT7 siRNA group was remarkably declined compared with the LN18 group (P<0.01 and P<0.01) (Fig. 5C and D). These findings suggest that actin polymerization and cell migration of SEPT7 siRNA-induced are reversed by cofilin inhibition and are cofilin-dependent.