Glioblastoma (GBM) is the most aggressive and lethal brain malignancy which is commonly referred to grade IV astrocytoma classified by World Health Organization (WHO). Despite there have great advancement in radiotherapy, chemotherapy, and surgical treatment, the median survival of GBM patients is merely about 14 months [
B-cell lymphoma 6 (BCL6), one of zinc finger transcriptional factor, works as a critical regulator in germinal center response. And this gene is also a key prooncogene of human B-cell lymphomas which participants in regulating the cell proliferation, differentiation and apoptosis of B and T cells [
In this study, we identified the high expression of BCL6 in glioma tissues and cell lines, and then we investigated the role of BCL6 expression in regulation of glioblastoma proliferation, migration, invasion, and chemosensitivity in vitro. In addition, we explored the underlying molecular events of BCL6 action in glioblastoma cells.
In this study, we collected twelve glioma tissue samples including six high-grade gliomas and six low-grade gliomas, and six nonmalignant brain tissues were obtained from Tianjin Huanhu Hospital. The six nonmalignant brain tissues which obtained from patients with traumatic brain injury were used as control. A protocol to use patient samples was approved by the ethics committee of Tianjin Huanhu Hospital and informed consent was obtained from each patient according to the Declaration of Helsinki.
Human GBM U87, A172, SNB19, U251, LN229, and LN308 cell lines were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) was used to incubate the cells, and the solutions were placed into a cell incubator for culture at 37°C with 5% CO2.
All plasmids were obtained from Gima Biol Engineering Inc. (Shanghai, China). Plasmid vectors pGPU6/GFP/NEO-shBCL6 contained a specific shRNA sequence according to the manufacture’s protocol. The pGPU6/GFP/Neo encoding nonspecific sequences were used as negative controls. And cells were transfected with plasmid vectors mediated by Lipofectamine 2000 (Invitrogen, USA) following the manufacturer’s directions.
Total RNA was extracted from tissue samples and glioblastoma cell lines with Trizol (Invitrogen, California, USA) according to the manufacturer’s instructions. The total RNA was reverse-transcribed by cDNA Reverse Transcription Kit (Invitrogen, California, USA) following the manufacturer’s protocol to get cDNA. To quantify expression of BCL6 mRNA, we used the Power SYBR green PCR master mix (Applied Biosystems, Carlsbad, USA) to detect BCL6 mRNA levels with a LightCycler 480 II PCR machine (Roche). GAPDH was measured as an internal control. The following primer pairs were used in this study: BCL6: 5’-CCAGCCACAAGACCGTCCAT-3’ (forward)5’-CTCCGCAGGTTTCGCATTT-3’ (reverse); GAPDH: 5’-TGCACCACCAACTGCTTAGC-3’(forward)5’GGCATGGACTGTGGTCATGAG-3’ (reverse).
Cell viability and proliferation were measured by CCK8 kit and colony-formation assay was performed. U87 and U251 cells were seeded in 96-well plates and cultured with DMEM medium (100
Cell apoptosis assays and cell cycle assays were performed at 48h after transfection
For glioma tissues, a protein extract reagent (Bioteke Corporation, Beijing, China) was used to extract the protein following the manufacturer’s directions. Cells seeded on six-well plates were washed with ice-cold PBS buffer and then homogenized by RIPA buffer supplemented with 1% protease inhibitors (Roche, Basel, Switzerland). After centrifugation, BCA Protein Assay Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used to determine the concentration of supernatant protein. And then the isolated supernatant protein was added with sample buffer and boiled at 95°C for 5 min.
Total proteins were separated by SDS-PAGE at 80V for 3h and then transferred to a PVDF membrane (Millipore, Bedford, MA, USA) for another 2h. Afterwards, the membranes were blocked by 5% skim milk in TBST buffer for 2h and incubated with primary antibodies at 4°C overnight. The primary antibodies included anti-BCL6, anti-BCL2, anti-Bax, anti-cyclin D1, anti-p21, anti-MMP2, anti-MMP9, anti-AKT, anti-p-AKT, anti-ERK, anti-p-ERK, and anti-GAPDH. (CST, Massachusetts, USA). Membranes were then washed by 1% TBST for three times and incubated with secondary antibodies for 1h. Finally, proteins were visualized by the ECL procedure (Millipore, Bedford, MA, USA) and were analyzed by Image J software to get the gray intensity of bands. Each experiment was performed in triplicate and repeated at least thrice.
We used Transwell (Corning, Cambridge, USA) to determine the effect of BCL6 on glioblastoma cells migration and invasion in vitro. To evaluate the invasive capacity of cells, Matrigel (BD Biosciences, FranklinLakes, NJ, USA) was used to cover the membrane to form a matrix barrier. At 24h after transfection, cells were seeded on the upper chamber with or without a Matrigel-coated membrane at a density of 5.0 × 103 cells/well in serum-free medium. The lower chamber was filled with 10% FBS as the chemoattractant. After 24 h incubation at 37°C, cells on the upper surface of the membrane were removed by wiping with a cotton swab, and cells on the lower surface were fixed with ethanol and stained with 1% crystal violet (Sigma-Aldrich, St Louis, MO, USA). And then the staining cells were photographed at 200× under a Nikon-TE2000 U inverted fluorescence microscope and counted by ImageJ software.
Wound-healing assay was preformed to assess capacity for tumor cell motility. Transfected cells were seeded into 6-well plates and grow to reach confluence. And then the cell layer was scratched using a plastic tip and incubated in DMEM with 10% FBS at 37°C for 72 h. Photos were taken at 0, 24, 48, and 72 h under microscope to measure the wound-healing after scratched. The wound gap percentage was determined by the ratio of gap width at each time point to the wound width at 0 h. Each experiment was carried out in triplicate, and three random fields of each well were recorded.
U87 and U251 cells were seeded in 96-wells plate overnight and transfected with scramble or shBCL6, and then treated with TMZ (Sigma, St Louis, USA) in different concentrations for 48h. CCK8 assay was performed to measure the cell survival. In addition, cell viability was measured at 0, 24, 48, and 72 h after TMZ (150
All data are presented as the mean ± standard deviation (SD) values and were analyzed with the GraphPad Prism 6.0 (CA, USA). One-way ANOVA or Student’s t-test was used for Statistical analyses. A p value of less than 0.05 was considered statistically significant.
To identify the expression of BCL6 in glioma, we detected its levels in tissues and cell lines. The BCL6 expression in glioma was higher than in normal brain tissues, and the mRNA level of BCL6 significantly elevated in the high-grade glioma samples compared with the low-grade samples (Figure
Expression of BCL6 mRNA and protein in human glioma tissues and glioblastoma cell lines. (a) The relative expression of BCL6 mRNA in 6 nonmalignant brain tissues and 12 glioma tissues was analyzed by RT-PCR. Data were normalized to the mean of mRNA in normal tissues. (b) The relative expression of BCL6 mRNA in glioblastoma cell lines (U87, A172, SNB19, U251, LN229, and LN308) was analyzed by RT-PCR. Data were normalized to the mean of mRNA in normal tissues. (c) The relative expression of BCL6 protein in nonmalignant brain tissues and glioma tissues was analyzed by western blot. Relative expression was calculated with respect to the first normal tissue. (d) The relative expression of BCL6 protein in glioblastoma cell lines by western blot. Relative expression was calculated with respect to the mean of protein in normal tissues. (e) Transfection efficiency of shBCL6 plasmid was confirmed by real-time PCR and western blot U87 and U251 cells. Relative expression was calculated with respect to the control group without treatment (
To investigate the role of BCL6 in progress of malignant gliomas, we reduced BCL6 expression by transfecting shBCL6 plasmid into the human glioma cell lines U87 and U251. Transfection efficiency was confirmed by real-time PCR and western blot analysis of BCL6 levels in the transfected glioma cells. BCL6 protein was decreased by 59.7% in U87 cells and 41.3% in U251 cells which were transfected with shBCL6 plasmid (Figure
We performed the cell viability assay and colony-formation assays to observe the effect of BCL6 on the glioma cell proliferation. The results showed that the growth of glioma cells transfected with shBCL6 was effectively inhibited compared with that of controls (Figures
The influence of BCL6 on the proliferation capacity, cell cycle progression, and cell apoptosis rate of glioblastoma cells. (a) CCK8 assay detected that cell proliferation vitality was inhibited in glioblastoma cells after BCL6 knockdown in U87 and U251 cells. (b) The result of clone formation assay showed that cell proliferation capacity was reduced after BCL6 knockdown U87 and U251 cells. Relative expression was calculated with respect to the control group without treatment. ((c) and (d)) The result of flow cytometry analysis of cell cycle and apoptosis in U87 and U251 cells transfected with shBCL6 or scramble sequences (
As shown in the Figure
Tumor cells with BCL6 knockdown resulted in significant reduction of cell migration (Figure
The influence of BCL6 on cell migration and invasion capacity. ((a) and (b)) Transwell assay was preformed to detect the migration and invasion capacity of glioblastoma cells after BCL6 knockdown. (c) Wound-healing assay was used to analyze the cell migration capacity of glioblastoma cells after BCL6 knockdown (
Western blot was performed to investigate the effect of BCL6 on the malignant behavior of tumor cells in protein level. These related protein levels changed along with the knockdown of BCL6 (Figure
The influence of BCL6 on molecules related to malignant behaviors of glioblastoma cells. (a) The western blot results of BCL2, Bax, cyclin D1, P21, MMP2, and MMP9 in indicated cells. GAPDH was used as reference. (b) The protein levels of t-ERK, p-ERK, t-AKT, and p-AKT in indicated cells. Relative expression was calculated with respect to the control group without treatment (
To further investigate the mechanism related to BCL6 promoting malignant phenotype of glioma, we detected the protein level of t-ERK, p-ERK, t-AKT, and p-AKT. Although there was no significant difference in t-ERK and t-AKT level between the shBCL6 group and the control groups, the results showed that knockdown BCL6 could inhibit the phosphorylation of ERK and AKT (Figure
The U87 and U251 cells were transfected with shBCL6 or scrambled and then treated with different concentrations of TMZ. As shown in Figure
Reduction of BCL6 increases chemosensitivity of Glioma Cells to Temozolomide. (a) The cells transfected with scrambled shRNA or shBCL6 were treated with TMZ at different concentrations, and cell survival was measured by CCK8 assay. Data were normalized to the Scrambled group with 0
As a human protooncogene, BCL6 was originally found in malignant lymphomas which acted as a regulator of the development and growth of B-lymphocyte [
Currently, it is unclear that the reason for BCL6 upregulated in cancer tissues, the constitutive expression caused by translocation might partly explain the overexpression of this gene in cancer tissues [
The function of BCL6 in cell proliferation and apoptosis has been explored for a long time with multiple molecules involved. Our research found that downregulation of BCL6 significantly reduced the expression of cyclin D1 and BCL2 while it upregulated the cyclin-dependent kinase inhibitor p21 and Bax. BCL6 was reported to induce the expression of cyclin D1 to cover the senescence response downstream of p53 by sequestering p21, but it could not repress p53-p19 pathway in senescence [
BCL6 exhibits its proinvasive ability in ovarian cancer [
The present research showed that knockdown BCL6 downregulated the expression of p-AKT and p-ERK. Both AKT and ERK are involved in receptor tyrosine kinase (RTK) pathway which is one of pathways emphasized in genomic analysis of GBM [
Furthermore, the glioblastoma was insensitive to chemotherapeutic drug, which led to chemotherapy failure and had a poor prognosis. In our study, we found that the reduction of BCL6 expression increased chemosensitivity of glioma cells to TMZ, and the apoptosis rates were upregulated in the reduction of BCL6 expression with TMZ group. Thus, BCL6 restoration approach may offer a new strategies and tactics to reverse or overcome multidrug resistance and hence to enhance the efficacy of TMZ treatment in glioma is one of the significant mission.
Our results show that BCL6 is expressed at a high level in glioma when compared with nontumor brain tissues. In addition, downregulation of BCL6 could inhibit proliferation, migration, and invasion, promote apoptosis, and induce cell cycle arrest via the AKT and MAPK pathway. Also, reduction of BCL6 increases chemosensitivity of glioblastoma to temozolomide. Thus, the identification of BCL6 may help us to understand the potential molecular mechanisms of the initiation and progression of glioma and provide us with a new prognostic marker for the management of glioma.
The data used to support the findings of this study are available from the corresponding author upon request.
All authors declare that they have no conflicts of interest.
Wen Song and Zhenling Wang contributed equally to this work.
This study was supported by Tianjin Health and Family Planning Commission (14KG118) and National Natural Science Foundation of China (81501035).