Background
Gliomas are the most common form of primary brain tumor in the adult central nervous system [
1]. Although recent studies showed progress in both diagnostic modalities and therapeutic strategies, glioma remains one of the deadliest human cancers. The five-year survival rate of patients with glioma is the lowest among those of all cancers [
2,
3]. The alternative treatment for glioma is limited due to the unclear pathophysiological mechanisms underlying the development of this disease. Therefore, understanding the molecular mechanism of the development and progression of glioma will shed light on strategies for accurate diagnosis, early intervention, and effective therapies.
Transcription factors (TFs) play important roles in the transcriptional networks that regulate gene expression, and misregulation of these TFs can result in the acquisition of tumor-related properties [
4]. Differentially expressed TFs in glioblastoma, and their downstream gene targets may be potential therapeutic biomarkers of glioblastoma [
5]. The MYBL2 gene, which is also known as B-MYB, is a member of the myeloblastosis family of transcription factors, first identified as cellular homologues of the
v-myb oncogene that is known to cause leukemia in chickens [
6]. MYBL2 of proliferative cells is crucial for the regulation of proliferation and differentiation, and also has a vital role in guiding cell cycle progression [
7]. Meanwhile, MYBL2 amplification or overexpression has been observed in cancers such as myeloid leukemias (AML) [
8], hepatocellular carcinoma [
9], breast cancer [
10], and it is currently used as a marker for poor prognosis in colorectal carcinoma [
11]. However, it is still not clear about the role of the MYBL2 gene in glioma.
FoxM1 is a member of the Forkhead box (Fox) transcription factor family, which has been shown to be over-expressed in various cancers and studies have shown that alterations in FoxM1 signaling were associated with carcinogenesis. FoxM1 is substantially elevated in several aggressive human carcinomas and can contribute to oncogenesis in many tissue types, including breast [
12], hepatocellular [
13], prostate [
14], lung [
15], and colorectal cancers [
16]. Aberrant FoxM1expression was found to be a common molecular alteration in malignant glioma [
17]. Moreover, it has been shown that higher expression of FoxM1 was associated with poor prognosis and radio resistance in glioma patient [
18‐
20]. Previous studies showed that FoxM1 was a key downstream gene of the Akt/FoxM1 signaling cascade [
21]. Another finding suggested that Akt/FoxM1 signaling played an impotent role in cervical cancer cell growth and treatment [
22]. However, the role and mechanism of Akt/FoxM1 signaling in the development of glioma is unknown.
Here, we have thoroughly investigated the expression levels of MYBL2 in glioma tissues and cell lines. And, we sought to determine whether Akt/FoxM1 signaling pathway is involved in regulating MYBL2 expression and whether these factors can predict the disease progression and prognosis of the glioma patients.
Methods
Patient samples
All samples, along with available clinical-pathological data, were obtained from were obtained from Xiangya Hospital of Central South University (Changsha, Hunan, China) between 2013 and 2014, with written informed consent. All pathological features were confirmed by experienced pathologists, and none of the patients received pre-operative anti-cancer treatment. All procedures were approved by the Ethics Committee Institute of Clinical Pharmacology, Central South University (Ethical Approval No. CTXY-1300041-3). These patients 79 had completed a follow-up time along with 48 months from the date of surgical resection. Overall survival (OS) was defined as a period time between the date of the initial surgical operation and death or the last follow-up.
We downloaded RNA-Seq gene expression data (Level 3) and clinical data from the TCGA data portal (
https://gdc-portal.nci.nih.gov/) as our other source of samples, and a total of 567 tumors having clinical data were profiled for class discovery and survival analysis [
23]. For overall survival (OS) and relapse-free survival (RFS) were searched in the glioma patient cohort in TCGA database using cBioportal (
http://cbioportal.org). The heat map and the correlation betweenMYBL2 and FoxM1 genes in the same patient cohort were further verified and analyzed using UCSC Xena t (
http://xena.ucsc.edu/). Moreover, the molecular functional network map of canonical pathways including coexpression, physical interaction, and predicted networks of FoxM1 analyzed by GeneMANIA (
http://genemania.org/).
Cell culture
Penicillin, streptomycin, trypsin-EDTA (ethylene diaminetetra acetic acid) were purchased from Beyotime (Beijing, China). The human glioma cell lines U251, U343, U87, T98G, and Hs683 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in and cultured in DMEM (Invitrogen, Shanghai, China), containing 10% fetal bovine serum (Gibco, Logan, UT, USA) and 100 U penicillin and streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.
Cell transfection
Three different interfering RNA (siRNA) for the specific inhibition of MYBL2 and FoxM1 expression and a negative control siRNA were synthesized by GenePharma Co., Ltd. (Shanghai, China). Exponentially growing untreated cells were plated 24 h before transfection. Plated cells were transfected with FoxM1 siRNA or MYBL2 siRNA at a final concentration of 50 nM, using 5 μL Lipo-RNAiMAX following the manufacturer’s instruction (Invitrogen, USA). After treatment, the cells were harvested and processed for further analysis. Human FoxM1 gene was inserted in pcDNA3.1 + HAvector and MYBL2 was inserted in GV230- GFP by Life Technologies (Shanghai Genechem, Co., Ltd., Shanghai, China) and the empty vector was used as the negative control. Hs683 cells transfected with FoxM1 and MYBL2 vectors with Lipofectamine 2000 according to the manufacturer’s information.
RNA extraction and quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from cultured cells or tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using Prime Script RT Master Mix (TaKaRa Biotechnology Co., Ltd., Dalian, China). Real-time quantitative PCR was performed using a standard SYBR Green PCR kit (Thermo). All reactions were conducted using the following cycling parameters: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, with a final extension at 60 °C for 60 s. GAPDH was used as an endogenous control. The gene expression was calculated using the ΔΔCt method. All data represent the average of three replicates. The primers used are listed in Table
1.
Table 1
Oligonucleotide primer sequences used in the qRT-PCR
MYBL2 | 5’-CTTGAGCGAGTCCAAAGACTG-3’ | 5’-AGTTGGTCAGAAGACTTCCCT-3’ |
FOXM1 | 5’-ATACGTGGATTGAGGACCACT-3’ | 5’-TCCAATGTCAAGTAGCGGTTG-3’ |
Akt | 5’-GACTACCTGCACTCGGAGAAG-3’ | 5’-TGTGATCTTAATGTGCCCGTC-3’ |
GAPDH | 5’-CCCATCACCATCTTCCAGGAG-3’ | 5’-CTTCTCCATGGTGGTGAAGACG-3’ |
β-actin | 5’-CATGTACGTTGCTATCCAGGC-3’ | 5’-CTCCTTAATGTCACGCACGAT-3’ |
Cell viability and proliferation assays
Cell viability and proliferation were measured by MTT assay after treatment. The identified cells were seeded in 96-well plates (6 × 103 cells/ well) and transfected with siRNAs. After culturing cell for an appropriate time, 50 μL of 5 mg/ml MTT (Sigma) was added to each well and cultured for 4 h. Then, the cell culture medium was replaced by 100 μL of dimethyl sulfoxide. After 2–3 h of incubation at 37 °C, the number of viable growing cells was estimated by measuring absorption at 570 nm wavelength and cell growth curves were determined according to the optical density value. The proliferation rate was calculated using the following formula: proliferation rate = survival rate = (OD test/ OD negative control) × 100%. All experiments were performed in triplicate and repeated at least three times.
Cell cycle analysis
Propidium iodide (PI) staining was used to analyze DNA content. Treated and untreated cells were harvested and labeled with PI by using previously described methods. Briefly, Cells in each group were washed with PBS for twice and centrifuged at 5000 rpm for 7 min to regulate the density as 1 × 106 cells/ well. Then, pre-cooling 70% ethyl alcohol was added for fixation overnight at −20 °C. On the next day, the fixed cells were washed with PBS, incubated with 400 μl PI/ RNase Staining Buffer (BD Company) at room temperature in the dark for 15 min. The cell cycle distribution was determined using a flow cytometer (Beckman Coulter, Brea, CA, USA). We then determined the percentage of cells in the G0/G1, S, and G2/M phases with the FlowJo software (Tree Star). The experiment was repeated for three times.
Apoptosis analysis
Apoptosis was assessed by Annexin V staining and flow cytometry analysis. Briefly, 3 × 105 cells were harvested, washed in PBS, and then analyzed by Annexin V/ propidium iodide staining according to the manufacturer’s protocol (FITC-Annexin V kit; BD Pharmingen, San Diego, CA). The stained cells were analyzed by flow cytometry.
U251 cells were seeded in 6-well plates (1.5 × 103 cells/ well); transfected with a non- silencing control siRNA, MYBL2 siRNA, or FoxM1 siRNA. After 15 days of incubation in the incubator, cell colonial forming amount was observed under the inverted microscope. And then, the cells were washed with PBS and stained with crystal violet, and visible colonies were counted.
Cell migration and motility
Cells were seeded in six-well plates (5 × 105 cells/well) and 24 h later were transfected with the control siRNA, MYBL2 siRNA (50 nM) or FoxM1 siRNA (50 nM). After culturing cell for an appropriate time, artificial wounds were gently made using a micropipette tip, and the cells were washed with PBS to remove floating cells and debris. The cells were then incubated in serum-free medium. Cells in the scratched area were imaged at 0 and 48 h using microscopy, and the distance traveled by cells at the leading edge of the wound at each time point was measured. The results were expressed as percent migration.
Transwell migration and invasion assays
Cell migration and invasion were assessed using a transwell assay. For migration assays, Matrigel (1:8) (BD Biosciences, Bedford, MA, USA) was diluted with serum-free DMEM, and the basement membrane of the upper chamber of the transwell was coated. The solution was kept at 37 °C for 1–4 h to transform the Matrigel aggregate into the gel. Treated cells were harvested and dilution with serum-free DMEM (5 × 10
5 cells/mL) 200 μL was added to a transwell insert (pore size, 8 μm; BD Biosciences, San Jose, CA, USA), and 600 μL containing 20% FBS was added to the lower chamber. Cells at each concentration were cultured in a 24-well plate in a 5% CO
2 incubator at 37 °C for 24 h [
24]. The culture medium in each well was then discarded, and the chamber was washed twice with PBS. And gently removing the cells in the upper chamber with a cotton swab, the cells on the underside of the membrane were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% cresyl violet, washed three times with PBS, and air-dried. Five fields (200 × magnification) were randomly selected for counting the number of migrated cells, and images were taken by using phase contrast microscopy.
Cell adhesion assay
Ninety-six-well dishes were pre-coated with 30 mg/L fibronectin solution (50 μL/well), then air-dried at room temperature overnight, and then rinsed with PBS and incubated with 3% heat-denatured BSA to block any uncoated areas. Cells were cultured in a 5% CO2 incubator at 37 °C for 1 h. The culture solution was then removed from the 24-well plate, and non-adherent cells were washed away three times with PBS. The remaining cells were fixed for 30 min with 4% paraformaldehyde, stained with cresyl violet for 15 min, and observed under an inverted microscope.
IHC
TMA slides were processed and stained manually as described previously. Formalin- fixed, paraffin-embedded sections were prepared for all tissues. Sections were deparaffinized in xylene and rehydrated through graded alcohol to water, and endogenous peroxidase activity was blocked by incubating the slides in 3% H2O2 in water for 30 min at room temperature. Sections were incubated in 1% BSA for 30 min then wiped off and dilution of MYBL2 (1:200, Santa Cruz) and FoxM1 (1:200, Santa Cruz) were applied to the slides and incubated overnight at room temperature. Subsequently, sections were incubated with secondary antibody for 2 h at room temperature, according to the manufacturer’s instructions. Negative control slides were processed in parallel using a non-specific immunoglobulin IgG (Sigma Chemical Co, St. Louis, MO, USA) at the same concentration as the primary antibody. Stained sections were observed under a microscope. Only fresh cut slides were stained simultaneously to minimize the influence of slide ageing and maximize repeatability and reproducibility of the experiment.
Immunofluorescence double staining
U251 cells were planted on glass slides in a 6-well at a density of 1 × 106 cells per well. Treated and untreated cells were fixed with ice-cold 4% paraformaldehyde for 30 min, permeabilised with 0.1% Triton X-100, and blocked in 2% gelatin in PBS at room temperature. Cells were then incubated with MYBL2 (Millipore, USA, 1:500) and FoxM1 (Cell Signaling Technology, USA, 1: 500) primary antibodies at 4 °C overnight. After being washed, the cells were incubated with secondary antibody (1:100) for 1 h at room temperature. The cell nucleus showed blue fluorescence (stained by DAPI). Images were obtained under a fluorescence microscope.
Western blot analysis
Cells from each group were collected, and whole-cell lysates were generated using RIPA lysis buffer (Abcam, Cambridge, UK). The protein concentration was detected by BCA method (U.S. Pierce Company). Total proteins were separated using 10% or 12% SDS-PAGE and then transferred onto a nitrocellulose membrane. The membrane was incubated with the primary antibody at 4 °C overnight, followed by a horseradish peroxidase-conjugated secondary antibody the next day for 2 h at room temperature. β-actin and GAPDH were purchased from (Sigma, USA). MYBL2 was purchased from (Millipore, USA). P-Akt and p-38 MAPK were purchased from (Santa Cruz Biotechnology, USA). SC79 (Akt activator) and MK-2206 (Akt inhibitor) were purchased from Selleck (Beijing, China). E-Cadherin(Wanleibio, China). Cyclins (CDK2, CDK6, CyclinD1, CyclinD3, CDK4, etc.), FoxM1, ZEB1, MMP9, MMP2, Vimentin and N- Cadherin were purchased from Cell Signaling Technology (Danvers, MA, USA).
Statistical analysis
Statistical significance between different groups was determined using t-tests for the MYBL2 and FoxM1 mRNA levels analysis and cell assay. Kaplan-Meier survival curves were used to compare survival rates. Univariate and multivariate Cox proportional hazard models were used to explore the associations between patient characteristics and biomarkers with outcomes. Statistical analyses were performed using SPSS19.0 software (IBM, Chicago, IL, USA). Data are shown as mean ± standard deviation. P-values <0.05 differences was considered statistically significant.
Discussion
In the present study, for the first time, we found that MYBL2 and FoxM1 are significantly associated with glioma progress; meanwhile, MYBL2 is interacted with radiotherapy for glioma survival. We also demonstrated the existence of a significant association between MYBL2 and FoxM1 expression in glioma. Furthermore, our results suggested that MYBL2 was downstream of the Akt/ FoxM1 signaling pathway.
Recently, studies found that transcription factors (TFs) and the transcriptional network play important roles in brain tumors progress [
25]. In the present study, two transcription factors MYBL2 and FoxM1 emerge as synergistic initiators and master regulators of glioma progress and transformation. We identified both FoxM1 and MYBL2 in high-grade glioma tissues were much higher than in normal brain tissues and low-grade glioma tissues. Either or both over expression of MYBL2 and FoxM1 were associated with poor prognostic.
The postoperative radiotherapy is an established standard treatment for glioma patients [
26]. However, glioblastoma is highly resistant to radiotherapy, which is a common problem with current anticancer therapies [
27]. So having an individualized radiotherapy plan based on each patient’s radio sensibility is necessary for increasing the treatment efficacy. Thus, the radio sensibility biomarker(s) can be very useful in glioma radiotherapy. The role of FoxM1 in radiotherapy has been reported in GBM [
19,
20,
28], but relatively little is known for MYBL2. In this study, we showed that MYBL2 is interacted with radiotherapy for glioma survival. GBM patients, those with MYBL2 high levels without radiotherapy had a significantly higher death risk than those with radiotherapy. Together, these findings further corroborate the rationale of MYBL2 and FoxM1 targeting in combination with irradiation.
Cell cycle progression and epithelial-mesenchymal transition (EMT) are key steps for tumor progress. Previous research had shown that MYBL2 and FoxM1 were both important cell cycle proliferation factors and might collaborate to induce mitosis [
29,
30]. To identify the molecular mechanism for the effects of MYBL2 and FoxM1 in glioma progress, we investigated the role of MYBL2 and FoxM1 in cell cycle progression and EMT. The results showed that knockout of MYBL2 and FoxM1 induced a G2/M phase arrest by down-regulation of cyclin B and cyclin D, but up-regulation of P21, P27 and CDK6. In addition, silencing of MYBL2 and FoxM1 down regulated the protein levels of N-cadherin and Vimentin but increased the levels of E-cadherin and ZEB1. These data indicated that MYBL2 and FoxM1 regulators of glioma progress and transformation by inducing cell cycle proliferation and EMT.
The BMYB-FoxM1 complex frequently observed and played an impotent role in cancers with poor prognosis and thought to promote cancer progression by up regulating the expression of mitotic genes [
31,
32]. Further study found that MYBL2 is required as a pioneer factor to enable FoxM1 binding to G2/M gene promoters [
29]. But, another report showed that a direct transcriptional regulation of FoxM1 by MYBL2, and a feedback loop between the latter and c-Myc, may be governing the replication machinery in ESCs [
29,
30]. Consistent with these results, we found that a strong correlation of the co-expression of FoxM1 and MYBL2 were observed in patients with gliomas. To further illuminate the relationship of MYBL2 and FoxM1 in glioma. We knocked down of MYBL2 and FoxM1 by siRNA in glioma cells and found that down-regulation of FoxM1 significant reduced MYBL2 protein expression; while down regulation of MYBL2 did a little change of FoxM1 expression. These results at least partially suggested that MYBL2 was a target of FoxM1 in glioma cells. But additional metadata is required to identify whether MYBL2 expression crucially regulated by FoxM1 through direct interaction with the MYBL2 promoter.
Studies have shown Akt pathway regulate various cell functions, such as angiogenesis, migration and invasion in glioma [
33,
34]. Moreover, it is showed that FoxM1 is a key downstream gene in the Akt signaling cascade [
21,
33]. In gastric cancer, Akt/FoxM1 signaling has been reported played an important role in chemotherapy [
21]. Wang et al. [
35] showed that Akt/FoxM1 axis was downstream of CXCL12 and took part in promoting GBM cell invasion. What is interesting is that some researchers reported that there is a positive regulatory feedback loop between FoxM1 and the PDGF/Akt signaling pathway, and the loop promotes breast cancer tumorigenesis [
36]. However, another research reported that blocking the Akt pathway by Akt-specific kinase inhibitor did not significantly alter FoxM1B transcriptional activity [
37]. Herein, we demonstrated that if Akt can regulate MYBL2 and FoxM1 expression in glioma cells. By knocking down p-Akt expression with Akt inhibitor lowered both FoxM1 and MYBL2 expression, and the activator elevated the two genes expression. Therefore, MYBL2 may be downstream of the Akt/ FoxM1 signaling pathway. Moreover, more studies are needed to see if the feedback loop of FoxM1 and Akt signaling pathway plays a role in MYBL2 expression in glioma.