Kank1, Bax, Bcl-2, Caspase-3, Caspase-9, CDK4, CDK6 and Cyclin D1 antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA). DMEM culture medium was from Sigma Co. (St. Louis, MO, USA). Fetal calf serum was from Gibco (Grand Island, NY, USA), siRNA and plasmid were synthesized by GenePharma Co., Ltd. (Shanghai, China), RT-PCR kit [Takara RNA PCR kit (AMV) Ver.3.0] was from Takara Biotechnology Co., Ltd. PI was from Sigma. The Annexin V-fluorescein isothiocyanate (FITC) kit was from Bio-Rad (Hercules, CA, USA).

Human brain glioma U87 and U251 cells (ATCC, Manassas, VA, USA) were cultured in the DMEM culture medium containing 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin, and then cultured in the incubator containing 5% CO₂ at 37°C.

Human brain glioma cells were inoculated in a 96-well culture plate with the density of 1×10⁵/ml and used in treatments when the cells grew to log phase. Six double wells were set for each experimental group. Each well was added with 20 μl of MTT (5 mg/ml) and cultured in CO2 incubator for 4 h before the culture solution was discarded. DMSO (150 μl) was added to each well at room temperature with oscillation for 10 min and a microplate reader was used for analysis.

Human brain glioma cells were inoculated in a 6-well culture plate with the density of 5×10⁵/ml. Transfection was carried out when the cells grow to higher than 70% confluence. Negative oligonucleotides was used as the control group. The culture medium was replaced to medium without serum or antibiotics, siRNA and Lipofectamine 2000 was added, respectively, adjust the preset concentration following the instructions of the transfection reagent. The culture plate was placed in CO₂ incubator for 4–6 h after siRNA transfection, and the culture continued using medium with serum. Following plasmid transfection the transfected cell line was screened. Resistant monoclone was established 3 weeks after the screening, the selected monoclone was expand in culture to establish the overexpressing Kank1 brain glioma cell line. RT-PCR and western blot analysis were performed.

Total RNA was extracted from the brain glioma cells referring to the instructions from RNAiso™ Plus (Takara, Japan). After determining total RNA concentration, RT-PCR reagent (Takara) was used for RT-PCR. Kank1 and β-actin gene primer were designed and synthesized by Invitrogen. Kank1 gene forward primer, 5′-GCACCCTGTCGTCTATCAACTC-3′; reverse primer, 5′-CTGCTGATTGGCTTTCCTTCT-3′. β-actin gene forward primer, 5′-CTGGGACGACATGGAGAAAA-3′; reverse primer, 5′-AAGGAAGGCTGGAAGAGTGC-3′. PCR reaction, 50 μl at the following reaction conditions: 94°C for 2 min, 94°C degeneration for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 30 sec, for a total of 31 cycles. The PCR product was electrophoresis with 1.5% agarose gel, then scanned and analyzed with gel imaging system (G: BOX Chemi XR5; Syngene, Cambridge, UK).

Trypsin was used to digest and collect the treated brain glioma cells, and the cells were suspended, with PBS to prepare a single cell suspension. Annexin V and PI staining fluid were added, respectively, in accordance with the Annexin V-fluorescein isothiocyanate (FITC) kit (Bio-Rad). Staining followed for 15 min at room temperature before the analysis with flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) for apoptosis.

Cells were cultured in a 6-well culture plate with cover glass coated with polylysine, then fixed with 4% paraformaldehyde for 30 min, and washed with PBS 3 times. Hoechst 33342 staining (5 μg/ml) fluid was added for 10 min incubation at 37°C, then wash with PBS 3 times. Observation and photography were performed under a fluorescence microscope.

Mitochondrial membrane potential is one of the indicators to measure the mitochondrial function. The change of mitochondrial membrane potential was analyzed as previously described (15). The results were analyzed with Cell Quest™ analysis software (Becton-Dickinson, USA).

Trypsin was used for digestion and collection of the treated brain glioma cells, and the cells were fixed with 70% ethanol at 4°C overnight, then washed with PBS twice. PI synthetic staining solution (1 ml) (containing 10 μg of RNase and 5 μl of Triton X-100) was added for 30 min at 4°C in the dark before analysis with flow cytometry (Becton-Dickinson) for apoptosis.

The brain glioma cells, of the experimental groups, were washed with PBS, and 2 ml of protein lysis solution (Sigma) was added for cell lysis. The protein concentration was detemined. The protein was separated on a 10% SDS-PAGE gel. The proteins were transferred to PVDF membrane with a semidry method and sealed with 5% skim-mild powder. The first antibody was applied for 2 h before membrane bleaching with TBST. The second antibody was applied for 2 h. Chemiluminesence was used for X-ray exposure imaging and strip scanning. Gray analysis was conducted using β-actin as the reference standard.

The experimental data were analyzed with SPSS 17.0 statistical software useing t-test and variance analysis. p<0.05 was considered to indicate a significant statistical difference.

RT-PCR and western blot assay were used to analyze the expressions of Kank1 gene and protein in U251 cells, U87 cells, HK-2 cells and GES-1 cells. It was found that the expressions of Kank1 gene and protein in U251 cells and U87 cells were both downregulated as compared with those of HK-2 cells and GES-1 cells, and the downregulation was the most obvious in U87 cells. The above results meant that the expression of Kank1 gene and protein was low in U87 cells and U251 cells of human brain glioma cells (Fig. 1).

In this experiment, U87 cells and U251 cells were selected to analyze the impact of silencing the Kank1 gene on the cell proliferation capacity with MTT method so as to investigate the impact of the Kank1 gene on the growth of human brain glioma cells. It was found that the proliferation capacity of U87 and U251 cells increased drastically after transfection of the Kank1 siRNA for 48 h as compared with the control group and the control group of transfection negative oligonucleotides. This indicated that silencing Kank1 gene could significantly promote the proliferation capacity of human brain glioma cells (Fig. 2).

In order to further confirm the impact of Kank1 gene on the growth of human brain glioma cells, we successfully built and transfected Kank1 plasmid to upregulate the expression level of Kank1 gene in human brain glioma cells and used the MTT method for analysis. It was found that the proliferation capacity of U87 cells and U251 cells reduced drastically after upregulating Kank1 gene expression level as compared with the control group and the control group of transfection negative oligonucleotides. This indicated that upregulating Kank1 gene expression could clearly inhibit the growth of human brain glioma cells (Fig. 3).

In order to investigate whether Kank1 gene is relevant to the apoptosis of human brain glioma cells, we upregulated the Kank1 gene expression level and used Annexin V-FITC/PI double labeling method to analyze the apoptosis rate. It was found that the apoptosis rate of U87 cells and U251 cells increased drastically after upregulating Kank1 gene expression level as compared with the N-control group (Fig. 4A). The western blot assay indicated that the expression level of Procaspase-9 and -3 proteins declined significantly (Fig. 4B and C). We used Hoechst 33342 staining for observation under the fluorescence microscope and found non-uniform agglutination of U87 cell chromatin with margination and pyknosis, or nuclear dense staining and formation of apoptosis bodies after upregulating Kank1 gene expression (Fig. 4D). The results indicated that upregulating Kank1 gene may result in the apoptosis of human brain glioma cells.

In order to investigate the relevant molecular mechanism of upregulating Kank1 gene-induced apoptosis of human brain glioma cells, we tested the change in the mitochondria membrane potential. It was found that upregulation of the Kank1 gene resulted in reduction of the mitochondria membrane potential (Fig. 5A). Additionally, we analyzed the mitochondria pathway apoptosis-relevant proteins Bax, Bcl-2 and cytochrome C via western blot assay. It was found that upregulating Kank1 gene resulted in an increase of Bax level of mitochondrion and a decline of Bcl-2 and cytochrome C level. However, the levels of intracytoplasm Bax, Bcl-2 and cytochrome C were the opposite to those of the mitochondrion (Fig. 5B–E). Our results indicated that upregulation of the Kank1 gene lowered the mitochondria membrane potential to promote the release of Bax and Bcl-2 as well as the release of cytochrome C into the cytoplasm so as to cause the apoptosis of human brain glioma cells.

In order to further explore the mechanism of Kank1 gene-inhibited proliferation of human brain glioma cells, we analyzed the change in the cell cycle by flow cytometry. The ratio of human brain glioma cells in phase G0/G1 was much higher than that of the control group of transfection negative oligonucleotides after upregulation of the Kank1 gene (Fig. 6A). Also, the expression levels of cell cycle proteins CDK4, CDK6 and Cyclin D1 declined greatly after upregulation of the Kank1 gene (Fig. 6B and C). Our results show that upregulation of the Kank1 gene inhibited proliferation of human brain glioma cells possibly by blocking the cells in phase G0/G1 via downregulating the expression of the proteins CDK4, CDK6 and Cyclin D1.