The protocol used in the present study was approved by the Ethics Committee of Central South University. Written informed consent was obtained from each participant. Forty-five glioma tissues (10 cases of WHO I, 13 cases of WHO II, 12 cases of WHO III, and 10 cases of WHO IV) were obtained from patients who underwent surgery from October 2011 to October 2012 at the Department of Neurosurgery, Xiangya Hospital of Central South University (Hunan, China), and 10 normal brain tissues from patients with cerebral trauma were used as controls. The human glioma U87 (WHO IV) and CHG-5 (WHO II) cell lines were obtained from the Cell Bank of Central South University. Dulbecco’s modified Eagle’s medium (DMEM), opti-MEM, fetal bovine serum (FBS) and Lipofectamine 2000 transfection agent were purchased from Invitrogen Life Technologies (USA). MTT was obtained from Sigma (USA). The bicinchoninic acid (BCA) protein assay kit was purchased from Pierce Biotechnology, Inc. (USA). PVDF membrane was obtained from Pall (USA). All siRNAs and antibodies were obtained from Santa Cruz Biotechnology, Inc. (USA). The Annexin V-FITC Apoptosis Detection kit was purchased from Biovision (USA). Matrigel was purchased form BD Biosciences (USA).

U87 and CHG-5 cell lines were cultured in DMEM containing 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. Cells were incubated at 37°C in a humidified incubator of 95% air and 5% CO₂.

The RACK1 mRNA expression in tissues or cell lines was detected by real-time RT-PCR (ABI 7500). Specific primers for different genes in this study were synthesized by BGI Company (Guangzhou, China). Specific primers for RACK1 were: sense, 5′-GAGCAAATGACCCTTCGT-3′ and antisense, 5′-GTAGTGCCCGTTGTGAGA-3′. Specific primers for Bax were: sense, 5′-CCCGAGAGGTCTTTTTCC GAG-3′ and antisense, 5′-CCAGCCCATGATGGTTCTGAT-3′. Specific primers for Bcl-2 were: sense, 5′-GGTGGGGTCAT GTGTGTGG-3′ and antisense, 5′-CGGTTCAGGTACTCAGTCATCC-3′. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used as a control (sense, 5′-GGCAGCCCAGAACATCATCC-3′ and antisense, 5′-GCCAGCCCAAGCATCAAAG-3′). All amplifications were performed in 3 parallel samples.

Cells were lysed by cold RIPA lysis, and the protein concentrations were determined using BCA protein assay kit. Then, proteins of 20 μg/lane were loaded on 12% SDS-PAGE to separate, and then electrophoretically transferred to PVDF membranes. Proteins on the membranes were then probed using primary antibodies according to the supplier’s protocol. Following incubation with secondary antibodies, results were visualized with peroxidase and an enhanced chemiluminescence system (Pierce Biotechnology, Inc.) and quantified by Quantity One software (Bio-Rad, USA).

Human glioma U87 and CHG-5 cells were seeded at a density of 10⁵ cells/well in 6-well plates and cultured in DMEM containing 10% FBS. After incubating at 37°C, 5% CO₂ for 24 h, U87 and CHG-5 cells were transfected with siRNA and Lipofectamine 2000 according to the supplier’s instruction. Briefly, 100 nmol siRNA and 5 μl Lipofectamine 2000 were diluted in opti-MEM to a final volume of 800 μl. After mixing for 20 min at room temperature, the siRNA/Lipofectamine 2000 mixture was added. Cells were incubated at 37°C 5% CO₂ for 6 h. Following incubation, the mixture was replaced with DMEM containing 10% FBS for 24 h.

Cell proliferation was determined by MTT assay. After 24 h post-transfection, the transfection medium in each well was replaced by DMEM medium containing 10% FBS used before, and was cultured for 12, 24, 36, 48 and 60 h. Then, the medium was replaced by 100 μl of fresh serum-free medium and cultured with 0.5 g/l MTT. Following incubation at 37°C for 4 h, the MTT medium was removed by aspiration and 50 μl of DMSO was added to each well. Following incubation at 37°C for a further 10 min, the A540 of each sample was measured using a plate reader.

Flow cytometry was used to determine the cell apoptosis with the Annexin V-FITC Apoptosis Detection kit. After 24 h post-transfection, cells were harvested and washed with cold PBS twice. Subsequently, 10⁶ cells were resuspended in 200 μl binding buffer supplemented with 10 μl Annexin V-FITC and 5 μl PI-PE, and incubated in the dark for 30 min. Then, 300 μl binding buffer was added followed by flow cytometry assay.

Cells (10⁵) of different groups in 200 μl serum-free DMEM were plated on the upper chamber plated on the top side of polycarbonate Transwell filter coated with Matrigel and incubated at 37°C for 24 h. Subsequently, cells inside the upper chamber were removed. Cells on the lower membrane surface were fixed in 1% paraformaldehyde, stained with 0.1% crystal violet and counted.

All animal experiments were approved by the Animal Care and Use Committee of Central South University. RACK1-specific shRNA lentivirus, or non-specific shRNA lentivirus, were purchased from Santa Cruz Biotechnology, Inc. Ten million U87 cells, which were without any treatment or infected with RACK1-specific shRNA lentivirus, or non-specific shRNA lentivirus, were injected subcutaneously into 8-week old nude mice (n=5), which were maintained under pathogen-limited conditions. The xenograft tumors were measured at 10, 15, 20, 25 and 30 days. We calculated the tumor volume according to V (mm) = D2 (mm²) × L (mm)/2, where D is the smallest perpendicular tumor diameters while L is the largest perpendicular tumor diameters. The animals were sacrificed at 30 days after implantation. All tumors were photographed and weighed.

Data are presented as means ± standard deviation (SD). SPSS 17.0 software was used to perform statistical analyses using a two-tailed Student’s t-test or one-way ANOVA. P<0.05 was considered to indicate statistically significant differences. All experiments were repeated at least 3 times.

To preliminarily investigate the relationship between RACK1 expression and glioma, we first examined the mRNA and protein expression levels of RACK1 in normal brain tissues, gliomas of different grades and 2 glioma cell lines. As shown in Fig. 1A and B, real-time PCR and western blotting results showed that the expression of RACK1 showed an increasing tendency with the malignancy of glioma. We further determined the expression of RACK1 in 2 human glioma cell lines, U87 (grade IV) and CHG-5 (grade II). As shown in Fig. 1C and D, real-time PCR and western blotting results demonstrated that RACK1 expression was also upregulated in U87 and CHG-5 cells compared to normal brain tissues (P<0.01). However, there was no difference of RACK1 expression between these 2 glioma cell lines (P>0.05). Accordingly, these data above indicate that the increased expression of RACK1 may be associated with the malignancy of glioma.

Due to the increased expression of RACK1 in glioma tissues and cell lines, we used RACK1-specific siRNA to downregulate the expression of RACK1 in U87 and CHG-5 cells to further investigate the role of RACK1 in glioma. After transfection, we first determined the mRNA and protein expression of RACK1 in U87 and CHG-5 cells using real-time RT-PCR and western blotting, respectively. As shown in Fig. 2, after transfection with RACK1-specific siRNA, the mRNA and protein levels of RACK1 in U87 and CHG-5 cells were effectively downregulated (P<0.01), when compared with those in the untreated (control) and the non-specific siRNA group (NC).

To further investigate the role of RACK1 in glioma cells in vitro, MTT assay was performed to determine the effect of siRNA-induced RACK1 downregulation on proliferation of U87 and CHG-5 cells. As shown in Fig. 3, MTT assay demonstrated that in RACK1-downregulated U87 and CHG-5 cells, the cell proliferation rate was lower when compared with controls (P<0.01). These results suggest that RACK1 promotes proliferation of human glioma cells.

We further investigated the effect of RACK1 downregulation on apoptosis of glioma cells. As shown in Fig. 4, in RACK1-downregulated U87 and CHG-5 cells, the cell apoptosis rate was much higher when compared with controls (P<0.01), suggesting that RACK1 may play an inhibitory role in the regulation of apoptosis of human glioma cells. We then examined the expression of apoptotic-related genes in each group. Real-time PCR assay showed that the mRNA expression of pro-apoptotic gene Bax was upregulated, while the mRNA expression of anti-apoptotic gene Bcl-2 was reduced, in RACK1-downregulated glioma U87 and CHG-5 cells, when compared with those in controls. These results indicated that RACK1 may suppress apoptosis of glioma cells in vitro through directly or indirectly regulating the expression of apoptotis-related genes, Bax and Bcl-2.

The alternations of cell invasion ability of human glioma U87 and CHG-5 cells were examined after transfection with RACK1-specific siRNA. As shown in Fig. 5, RACK1-downregulated cells showed decreased invasion ability when compared with controls (P<0.05). These data indicate that RACK1 promotes invasion ability of human glioma cells in vitro.

Bax is a pro-apoptotic gene, while Bcl-2 has an anti-apoptotic function. It has been well established that the ratio of Bax/Bcl-2 protein plays a crucial role in regulating cell apoptosis. Hence, we applied real-time RT-PCR to determine the mRNA expression of Bax and Bcl-2 in U87 and CHG-5 cells of each group. As shown in Fig. 6A, the relative mRNA level of Bax was significantly upregulated in U87 and CHG-5 cells after transfection with RACK1-specific siRNA, when compared with that in the control group (P<0.01). However, the relative mRNA level of Bcl-2 was decreased in U87 and CHG-5 cells after transfection with RACK1-specific siRNA, compared with that in the control group (P<0.01). These data partly explain the above findings that forced downregulation of RACK1 promotes the apoptosis of U87 and CHG-5 cells.

Src/Akt signaling pathway plays a crucial role in the regulation of survival, proliferation and migration of multiple types of cells. It has been reported that RACK1 induces colon cell apoptosis, partly by suppressing Src activity in Akt pathway. However, whether a similar molecular mechanism exists in glioma cells remains unknown. Thus, we determined the activity of Src/Akt signaling pathway in RACK1-downregulated U87 and CHG-5 cells. As shown in Fig. 6B, the phosphorylation levels of Src and Akt were much lower in U87 and CHG-5 cells after transfection with RACK1-specific siRNA, when compared with those in controls (P<0.05). These results indicate that the effects of RACK1 on the survival, proliferation, invasion of glioma cells may be involved in the regulation of the Src/Akt signal pathway.

To further investigate the role of RACK1 in vivo, a tumor xenograft animal model was conducted using U87 cells, in which RACK1 was successfully knocked down by lentivirus infection. After all animals were subcutaneously implanted with the infected tumor cells, the size of tumors in the RACK1-specific shRNA group was significantly smaller than that in the control and NC group (Fig. 7A). As shown in Fig. 7B, the average tumor weight in the RACK1-specific shRNA group was markedly lower than that in the control and NC group. Moreover, the tumor size in the RACK1-specific shRNA group was much smaller than that in the control and NC group (Fig. 7C). These results are consistent with those of the cell proliferation assay, indicating that forced downregulation of RACK1 significantly inhibits tumor growth, and that RACK1 plays a positive regulatory role in glioma growth in vivo.