Cell Mol Neurobiol DOI 10.1007/s10571-015-0237-z
ORIGINAL RESEARCH
CHD1L Regulates Cell Cycle, Apoptosis, and Migration in Glioma Jie Sun1 • Li Zhang2 • Hongyu Zhao1 • Xiaojun Qiu1 • Wenjuan Chen1 • Donglin Wang2 • Na Ban2 • Shaochen Fan2 • Chaoyan Shen1 • Xiaojie Xia1 Bin Ji1 • Yuchan Wang2
•
Received: 12 May 2015 / Accepted: 6 July 2015 Ó Springer Science+Business Media New York 2015
Abstract Chromodomain helicase/ATPase DNA binding protein 1-like (CHD1L) gene is a newly identified oncogene located at Chr1q21 and it is amplified in many solid tumors. In this study, we intended to investigate the clinical significance of CHD1L expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that CHD1L was overexpressed in glioma tissues and glioma cell lines. In addition, the expression level of CHD1L was positively correlated with glioma pathological grade and Ki-67 expression. Kaplan–Meier curve indicated that high expression of CHD1L may result in poor prognosis of glioma patients. Accordingly, suppression of CHD1L in glioma cells was shown to induce cell cycle arrest and increase apoptosis. In addition, knockdown of CHD1L significantly accelerated migration and invasion ability of glioma cells. Together our findings suggest that CHD1L is involved in the progression of glioma and may be a novel target for further therapy.
Jie Sun and Li Zhang have contributed equally to this work. & Bin Ji [email protected] & Yuchan Wang [email protected] 1
Department of Radiotherapy and Oncology, The Affiliated Hospital of Nantong University, Xisi Road No. 20, Nantong 226001, People’s Republic of China
2
Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College of Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
Keywords Migration
CHD1L Glioma Cell cycle Apoptosis
Introduction Malignant glioma, highly invasive primary brain tumor, is the most frequent type of primary adult brain neoplasms (Venkataramanaa et al. 2013; Wen and Kesari 2008). WHO classification based on the malignancy of the neoplasms ranges from grade I, corresponding to low-proliferative noninvasive tumors, up to grade IV, assigned to cytologically malignant, highly infiltrative, mitotically active, and necrosis-prone glioblastoma multiforme (GBM, malignant glioma) (Sims et al. 2015). Current standard of care includes maximal safe surgical resection, fractionated radiation, and systemic temozolomide chemotherapy (Zhuang et al. 2009). Despite advances in conventional treatment, the outcome for glioma patients remains almost universally fatal. This poor prognosis is mainly due to therapeutic resistance and tumor recurrence after surgical removal (Wang et al. 2015). Therefore, it is urgent to reveal the underlying main molecular mechanisms of this malignancy so as to develop more effective therapies and improve the disappointing outcome. The chromodomain helicase DNA binding protein 1-like (CHD1L) gene is a putative oncogene mapped to chromosome 1q21 (Cheng et al. 2013). The CHD family of proteins is ATP-dependent chromatin remodeling enzymes which combine chromodomains with SWI2/SNF2 ATPase/ helicase motifs and have DNA binding capability (Cheng et al. 2013). The clinical significance of amplification and overexpression of CHD1L has been evaluated in solid tumors, including HCC (Hyeon et al. 2013), ovarian carcinoma (He et al. 2012), colorectal carcinoma (Ji et al.
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2013), and bladder cancer (Tian et al. 2013). All these studies demonstrate that CHD1L is a novel biomarker for prediction of progression, prognosis, and survival. However, to the best of our knowledge, there is as yet no report demonstrating the function and mechanism of CHD1L in glioma. In this study, we further verified the high expression of CHD1L in glioma tissues and cell lines, and then, we analyzed its relationship with clinicopathological features and its prognostic value for glioma patients’ survival. Furthermore, we investigated the possible role of CHD1L in cell cycle, apoptosis, migration, and invasion of glioma. According to our research, it was reasonable to consider that CHD1L could be a novel therapeutic target for glioma.
Materials and Methods Patients and Tissue Samples For Western analysis, all fresh frozen human glioma tissue samples were stored at -80 °C immediately after surgical removal. While for immunohistochemical analysis, a total of 81 glioma specimens were obtained from the Department of Pathology, Affiliated Hospital of Nantong University from 2005 to 2011. None of the patients were treated with such preoperative therapies as immunotherapy, radiation, or chemotherapy. All patients were followed up for 1–60 months. For histological examination, all tissues were fixed in 10 % buffered formalin and embedded in paraffin for sectioning. The glioma samples collection was executed in accordance with the protocols approved by the Ethics Committee of the Affiliated Hospital of Nantong University. The main clinical and pathologic variables are shown in Table 1. Immunohistochemical Staining Surgically excised tissues were fixed with 10 % formalin and embedded in paraffin, and 4-lm-thick specimen sections were prepared on glass slides. Tumor tissue microarray blocks were deparaffinized using a graded ethanol series, and endogenous peroxidase activity was blocked by soaking in 0.3 % hydrogen peroxide. Then, the sections were processed in 10 mmol/L citrate buffer (pH 6.0) and heated to 121 °C in an autoclave for 3 min to retrieve the antigen. Hydrogen peroxide (0.3 %) was applied to block endogenous peroxide activity for 20 min after cooling. After rinsing in PBS (pH 7.2), the sections were incubated with mouse anti-human CHD1L antibody (diluted 1:50) and mouse anti-human Ki-67 antibody (diluted 1:400) for 2.5 h at room temperature. All slides were processed using the peroxidase–antiperoxidase method
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(Dako, Hamburg, Germany). After washing with PBS, the peroxidase reaction was visualized by incubating the sections with DAB (0.1 % phosphate buffer solution, 0.02 % diaminobenzidinetetrahydrochloride, and 3 % H2O2). After being rinsed in water, the sections were counterstained with hematoxylin. At last, the sections were dehydrated with graded alcohol and cover slipped. Immunohistochemical Analysis The immunostained sections were evaluated by three pathologists without knowledge of the clinical and pathological parameters of the patients in a blinded manner. For assessment of CHD1L and Ki-67 expression, at least five high-power fields were chosen randomly in each specimen, and more than 300 cells were examined to determine the mean percentage of signal-positive cells. The staining results were scored semiquantitatively. The percentage of cells was scored as follows: 1 (0–49 % tumor cells stained), 2 (50–74 % tumor cells stained), and 3 (75–100 % tumor cells stained). The intensity of staining was coded as follows: 1 (weak staining), 2 (moderate staining), and 3 (strong staining). Then, we multiplied the two scores and classified them into two groups: low expression (0–4.5) and high expression (4.5–9). As for statistical analysis of Ki-67 stain, a cutoff value was used to distinguish tumors with a low (\50 %) or high (C50 %) level of Ki-67 expression. Antibodies The antibodies used for Western blot analysis and immunohistochemistry were as follows: anti-CHD1L monoclonal antibody (Santa Cruz Biotechnology, USA), antihuman Ki-67 monoclonal antibody (Santa Cruz Biotechnology, USA), anti-human proliferating cell nuclear antigen (PCNA) monoclonal antibody (Santa Cruz Biotechnology, USA), anti-human cyclin D1,anti-human cyclin E polyclonal antibody (Santa Cruz Biotechnology, USA), p53, p21(Santa Cruz Biotechnology, USA), E-cadherin, b-catenin, N-cadherin, vimentin(Santa Cruz Biotechnology, USA),and anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibody (Santa Cruz Biotechnology, USA). Cell Cultures The human glioma cell lines U87MG, U251MG, U118MG, A172, and H4 were obtained from the cell library of the Chinese Academy of Sciences and were cultured in Dulbecco’s modified Eagle medium (DMEM) (GibCo BRL, Grand Island, NY, USA) with 10 % fetal bovine serum, 2 mM L-glutamine, and 100 U/mL penicillin–streptomycin
Cell Mol Neurobiol Table 1 CHD1L expression and clinicopathological variables in 81 glioma specimens
Variables
Total
v2 value
CHD1L expression Low score \5
P value
High score C5
Age (years) \50
47
20
27
C50
34
18
16
0.855
0.355
0.048
0.827
4.164
0.384
1.052
0.591
0.046
0.829
12.542
0.002*
21.287
0.001*
Gender Male
48
23
25
Female
33
15
18
Tumor location Frontal
23
9
14
Parietal
10
5
5
Occipital
11
4
7
Temporal Unknown
17 20
11 12
6 8
Extent of resection Biopsy
16
9
7
Total resection
28
12
16
Subtotal resection
37
20
17
Tumor size (cm) \4
17
9
8
C4
64
32
32
II
22
18
4
III
29
13
16
IV
30
10
20
Low expression
56
28
28
High expression
44
10
34
WHO grade
Ki67
Statistical analyses were performed by the Pearson v2 test * P \ 0.05 was considered significant
mixture (GibCo BRL) at 37 °C, and 5 % CO2. The medium was changed every 2–3 days, and cultures were split using 0.25 % trypsin. Western Blot Tissue and cell protein were promptly homogenized in a homogenization buffer containing 1 M TrisHCl pH 7.5, 1 % Triton X-100, 1 % Nonidet p-40 (NP-40), 10 % sodium dodecyl sulfate (SDS), 0.5 % sodium deoxycholate, 0.5 M EDTA, leupeptin 10 lg/mL, aprotinin 10 lg/mL, and 1 mMPMSF, and then centrifuged at 10,0009g for 30 min to collect the supernatant liquid. Protein concentrations were determined with a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). The total cellular protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter (PVDF) membranes (Millipore, Bedford, MA). After the membranes
were blocked in 5 % nonfat milk in TBST (150 mMNaCl, 20 mMTris, 0.05 % Tween 20) for 2 h, they were incubated with the primary antibodies overnight at 4 °C. Then, the membranes were washed with TBST for three times, 5 min each, and then horseradish-peroxidase-linked IgG as the secondary antibodies for 2 h at room temperature. The membrane was developed using the ECL detection systems. The experiments were carried out in three separate occasions. Transient Transfection Cells were grown in dishes until they reached 80 % confluence. The medium was replaced 6 h later with fresh medium for transfection. The CHD1L-siRNA and control-siRNA were purchased from Genechem (Shanghai). The CHD1L-specific siRNA target sequence: CHD1L-siRNA#1 was 50 -GTATTGGACATGCCACGAAA-30 , CHD1L-siRNA#2 was 50 -TAT TGGACATGCCACGAAA-30 , CHD1L-siRNA#3 was 50 -
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CAAGAGAAGGAGACTCATA-30 , and CHD1L-siRNA#4 was 50 -ACAAACTCTTGCAGCCATT-30 . Glioma cells were transfected with CHD1L-siRNA or control-siRNA according to the manufacture’s instructions. Cells were collected for Western blot, CCK-8, and flow cytometry assays after transfection for 24–48 h. Flow Cytometry Analysis of Cell Cycle and Cell Apoptosis To synchronize the cell cultures, we seeded U87 cells in 6-cm dishes in growth medium with 10 % FBS overnight. Then, the cultures were rinsed by PBS and changed to serum-free medium. After serum starvation for 72 h, the cells were released into cell cycle by addition of 10 % FBS. For flow cytometrical analysis of cell cycle distribution, cells were collected and fixed in 70 % ethanol over night at 4 °C, washed with PBS, and then incubated with 1 mg/mL RNase A for 30 min at 37 °C. Then, cells were stained with 50 mg/mL propidium iodide (PI) (Becton– Dickinson, San Jose, CA) in PBS and 1 % Triton X-100. The data were acquired using a BD FACSCAN flow cytometer (Becton–Dickinson, San Jose, CA) and analyzed using the Cell Quest Acquisition and analysis programs (Becton–Dickinson). U87 cells transfected with CHD1LsiRNA and control-siRNA were cultured for 48 h and harvested. Then, add 60 lL of MuseTMAnnexin V & Dead Cell Reagent (Part No. 4700-1485, 100 tests/bottle) to each tube and add 60 lL of cells in suspension to each tube. After incubated for 20 min at room temperature in the dark, the apoptosis assay was performed by MuseTM Cell Analyser (EMD Millipore corporation) according to the manufacturer’s instructions. Cell Proliferation Assays Cell growth was measured using a commercial Cell Counting Kit (CCK)-8 (Dojindo, Kumamoto, Japan) in accordance with the manufacturer’s instructions. Briefly, cells were plated into a 96-well plate at a density of 2 9 104 cells/well and grown overnight. Cell Counting Kit-8 reagent was added to a subset of wells for 2 h incubation at 37 °C, and the absorbance at the wavelength of 490 nm was read in an automated plate reader. The experiments were repeated at least three times. Plate Colony Formation Assay NC and transfection of CHD1L siRNA U87MG cells (2000 cells/plate) were cultured in 5 mL of DMEM supplemented with 10 % FBS and 800 mg/mL G418 in a 6-cm plate. After 14 days, colonies were washed with PBS, fixed with methanol for 30 min, and stained with crystal violet for
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30 min. Clearly visible colonies ([50 mm in diameter) were counted as positive for growth. Monolayer Wound Healing Assays U87 cells were seeded on 6-well plates at a density of 4 9 105 cells/well. Thirty-six hours after transfection, the cells were scratched using a sterile 10 lL micropipette tip, and washed three times with PBS. Then, cells were then cultured in complete DMEM medium for an additional 48 h. Photographs were taken by an inverted Leica phasecontrast microscope (Leica DFC 300 FX) at 0, 12, and 24 h time points. Trans-well Migration Assay Into the trans-well chambers (Corning, 8 lm pore size) were seeded 1 9 105 cells in the upper chambers in medium, and DMEM with 10 % FBS was added to the bottom chambers. After 24 h of incubation, the filters were stained with crystal violet. The number of migrating cells in 5 fields was counted under 9200 magnification, and the means for each chamber were determined. Statistical Analysis All the data were carried out by SPSS17.0 statistical package from SPSS Inc. (Chicago, USA). The relationship between the CHD1L expression and the clinical features was analyzed by Chi-square (v2) or Fisher’s exact test. Correlation analysis was also used. For analyzing the survival data, Kaplan–Meier curves were constructed, and the log-rank test was performed. Cox’s proportional hazards regression model was used for multivariate survival analysis, and the risk ratio and its 95 % confidence interval were recorded for every marker. Student’s test was used for comparison between groups. P values \0.05 were considered to be statistically significant.
Results CHD1L Overexpression in Glioma Tissues To identify whether CHD1L associated with glioma or not, we evaluated the expression level of CHD1L in seven fresh glioma tissues from Grade II to Grade IV by Western blot. We found that CHD1L was overexpressed in glioma tissues, and its expression was significantly higher in highgrade glioma tissues than in low-grade glioma tissues (Fig. 1a, b). Then, we also analyzed CHD1L expression by immunohistochemistry for 81 glioma samples in order to demonstrate the association of CHD1L with glioma
Cell Mol Neurobiol Fig. 1 The correlation between CHD1L and glioma grade, Ki67 and glioma outcome. a Western blot analysis of expression of CHD1L in samples of glioma tissues (grades II–IV). b The bar chart demonstrates the ratio of CHD1L protein to GADPH for the above by densitometry. The data are mean ± SD of three independent experiments. c Paraffin-embedded glioma tissue sections (including grades II–IV) were stained with antiCHD1L antibodies and antiKi67 antibodies followed by counterstaining with hematoxylin (SP 9400). The paraffin-embedded tissue sections were stained with no antibodies as negative control (SP 9400). d The relationship between CHD1L and Ki67. e Kaplan–Meier survival curves for high CHD1L expression versus low CHD1L expression in 81 patients with glioma. On the basis of mean CHD1L percentages, patients were divided into high CHD1L expressers and low CHD1L expressers. Patients in the high expression of CHD1L group had a significantly shorter overall survival (P \ 0.01)
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progression. As shown in Fig. 1c, CHD1L localized in the nucleus of glioma cells, and its expression increased as the malignant degree increased, which was consistent with Ki67 expression. Ki-67 was considered to be associated with cellular growth and proliferation (Yamashita et al. 2010). Furthermore, there was a positive correlation between CHD1L and Ki-67 expression in the examined glioma tissues (Fig. 1d). Relationship Between CHD1L Expression and Glioma Patients’ Survival The immunohistochemical results of 81 glioma specimens are summarized in Table 1. In short, CHD1L expression was apparently associated with clinicopathologic grades (P = 0.002) and Ki67 expression (P = 0.001). Little obvious correlation was observed between CHD1L expression and patient’s gender, age, tumor location, extent of resection, or tumor size in 81 glioma cases. When a multivariate Cox proportional hazard model was constructed, we found that WHO grade, the high Ki67 expression, and CHD1L expression were prognostic factors of overall survival (P = 0.010, 0.001, 0.041; Table 2). Kaplan–Meier survival curves indicated that up-regulation of CHD1L was significantly associated with poor overall survival (Fig. 1e). Combined these results together, we inferred that high expression of CHD1L could be a strong determinant of poor prognosis in glioma. Expression of CHD1L Promotes G1/S Phase Transition Since CHD1L was correlated with the expression of Ki-67 in glioma specimens, we proposed that CHD1L might play a role in cell cycle progression of glioma cells. Among the glioma cell lines, CHD1L was expressed highly in U87 and
Table 2 Contribution of various potential prognostic factors to survival by Cox regression analysis on 81 glioma specimens Characteristic
Hazard ratio
95 % CI
P value
Age
0.821
0.433–1.554
0.544
Gender
1.522
0.783–2.959
0.216
Tumor location
1.187
0.952–1.479
0.127
Extent of resection
0.891
0.543–1.462
0.648
Tumor size
0.756
0.326–1.755
0.516
WHO grade
2.225
1.213–2.225
0.010*
CHD1L expression
2.151
1.033–4.478
0.041*
5.180–387.666
0.001*
Ki67 expression
44.814
Statistical analyses were performed by the Cox regression analysis CI confidence interval * P \ 0.05 was considered significant
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Fig. 2 The expression of CHD1L and cell cycle-related molecules in c glioma cells. a, b The expression of CHD1L in five glioma cell lines (H4, A172, U118, U87, and U251MG) and protein levels of CHD1L were quantified by correcting the average pixel intensity for each band with that of GAPDH as an internal control for equal loading of protein samples. The data are mean ± SD. c, e After serum starvation for 72 h followed by the addition of medium containing 10 % FBS for the indicated times, flow cytometry quantitation of cell cycle progress in U87 cells. d, f After serum starvation for 72 h, followed by the addition of medium containing 10 % FBS for the indicated times, flow cytometry quantitation of cell cycle progress in U251 cells. g, h U87 cells were serum starved for 72 h, and cell lysates were prepared at the indicated time points after serum refeeding and analyzed by Western blot using antibodies directed against CHD1L, PCNA, cyclin D1, and GAPDH (a control for protein load and integrity). The bar chart demonstrates the ratio of CHD1L, PCNA, and cyclin D1 protein to GAPDH for each time point by densitometry. Mean ± SEM of three independent experiments. *, #, ^P \ 0.05, compared with control cell serum starved for 72 h (S72 h). S serum starvation, R serum release, SEM standard error of the mean
U251 (Fig. 2a, b). So in the following experiment, we used them as the main experimental cell lines. To certify that CHD1L was involved in the cell cycle, we built a serum starvation and releasing model. The U87 and U251 cells were arrested in G1 phase after serum starvation for 72 h, and they were released into cell cycle by addition of 10 % FBS. Then, cells were harvested at 0, 4, 8, 12, and 24 h. After serum re-addition, the U87 cells were released from the G1 phase (from 73.19 to 41.33 %) and re-entered the S phase (from 7.42 to 38.03 %), and the U251 cells were released from the G1 phase (from 87.77 to 56.45 %) and re-entered the S phase (from 8.5 to 30.83 %). Flow cytometry results showed that the transition of G0/G1-S in U87 and U251 cells increased gradually (Fig. 2c–f). Next, Western blot was used to analyze the expression of CHD1L during cell progression, as well as the proliferation markers such as PCNA and cyclin D1. We collected the U87 cell protein at different time points, and as expected, we found that CHD1L was gradually increased after serum re-addition. Meanwhile, the cell proliferation markers PCNA and cyclin D1 had a similar tendency with the CHD1L protein level (Fig. 2g, h). These results affirmed that CHD1L may be involved in the proliferation of glioma in a cell-cycledependent pathway. Knockdown of the Expression of CHD1L Inhibited the Proliferation of Glioma Cells To further study the biologic effect of CHD1L on cell cycle, we transfected CHD1L siRNAs and control-siRNA into U87 cells to knockdown endogenous CHD1L. We used four siRNAs directed against CHD1L expression to examine their interference efficiency. U87MG cell lines were transfected with four siRNA duplexes for 48 h; the
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Fig. 3 Silencing of CHD1L decreases glioma cell growth. a We analyzed CHD1L expression by Western blot after transfected with CHD1L-siRNA in U87 cells, while CHD1L-siRNA4 achieved the best down-regulation effect. The data are mean ± SEM. *P \ 0.05 compared with the control. b Western blot analysis showing the effect of CHD1L knockdown on the protein expression of p53, p21, cyclin E, and Cdk2. c The bar chart demonstrates the ratio of CHD1L protein to GADPH for the above by densitometry (P \ 0.05).
d Growth curves of U87MG cells transfected with the CHD1L siRNA or control-siRNA. e Flow cytometric analysis of cell cycle distribution 48 h later following control-siRNA and CHDIL siRNA4 transfection. f CHD1L knockdown suppresses plate colony formation. Equal numbers of U87-control-siRNA cell and CHD1L-siRNAU87 cells were seeded onto 60-mm plates. The cells were fixed and stained with Giemsa after 14 days; the number of colonies in CHD1LsiRNAU87 cells was less than that in the con-siRNA cells
CHD1L-siRNA4 resulted in around 50 % decrease in the expression of CHD1L on protein levels compared to the control-siRNA (Fig. 3a, c). Then, we used the controlsiRNA and the CHD1L-siRNA4 for the further research. Finally, we further proved the role of CHD1L in glioma cell cycle. Suppression of endogenous CHD1L in U87MG cells inhibited the cell viability effectively and the cell cycle by CCK8 assay and flow cytometry, and plate colony formation assays indicated the same effect (Fig. 3d–f). Furthermore, DNA content analysis using flow cytometry showed that si-CHD1L was able to inhibit the cell cycle at the G1/S checkpoint (Fig. 3e). The percentage of cells in the S phase was significantly reduced in the si-CHD1Ltreated cells compared with the control-si-treated cells. It was reported that in a transgenic mouse model, CHD1L could facilitate DNA synthesis and G1/S transition through
the up-regulation of Cyclins (A, D1, and E), CDK2, 4, and down-regulation of Rb, p27 (Kip1), and p53 (Chen et al. 2009b; Liu et al. 2012). In our study, the CHD1L expression was decreased obviously and it was in accordance with the up-regulation of p53 and p21, down-regulation of cyclin E and Cdk2 (Fig. 3b). As a result, these results suggested that CHD1L may regulate cell cycle through p53–cyclinE–Cdk2 pathway and it would promote proliferation of glioma cells, and its knockdown could suppress the proliferation of glioma cells.
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Loss of CHD1L Expression Induced Apoptosis in Glioma Cells The decrease of apoptosis is another major mechanism of an oncogene in cancer development; we presumed CHD1L
Cell Mol Neurobiol Fig. 4 Effect of CHD1L downexpression on apoptosis. a U87 cells were treated with controlsiRNA and CHD1L-siRNA4, respectively, and then assayed by MuseTM Cell Analyser according to the manufacturer’s instructions. b Western blot was used for the analysis of antiapoptosis Bcl-2 and active caspase 3 expression in controlsiRNA and CHD1L-siRNA4transfected U87 cells
might be related to apoptosis in glioma. We next examined the effect of CHD1L on glioma cells’ survival. A flow cytometry assay was used to investigate whether CHD1L induced apoptosis in glioma cells. From the results we could conclude that, compared with control-siRNA-transfected cell, there were more apoptotic cells in the CHD1LsiRNA#4-transfected cells (Fig. 4a). To further study the mechanism of CHD1L in glioma apoptosis, Western assay was performed to detect apoptosis-related genes such as active caspase 3 and Bcl-2 (which considered of antiapoptotic) (Fan et al. 2014; Lin et al. 2004; Takano et al. 2014; Yang et al. 2014). As expected, knockdown of CHD1L resulted in increased expression of active caspase 3, with concomitant inhibited expression of Bcl-2 (Fig. 4b). All these results suggested that CHD1L expression might contribute to tumorigenesis through its antiapoptotic effect. CHD1L was Associated with the Migration of the Glioma Cell Line Functional studies in vitro and in vivo showed that CHD1L contributed to tumor cell migration and invasion by increasing cell motility and inducing filopodia formation and epithelial-mesenchymal transition (EMT) (Chen et al. 2010). So we supposed that CHD1L perhaps was related to
the migration and invasion of glioma. In the wound healing assay, we could see that the migration rate of the CHD1LsiRNA4 group was slower than the control group, which indicated that the ability of migration in the siRNA4 group was decreased by knocking down the expression of CHD1L compared to the negative group (Fig. 5a). Meanwhile, results from the trans-well assay revealed that knocking down the expression of CHD1L inhibited the cell migration to the bottom chambers compared to the control and negative groups (Fig. 5b). As a result, we supposed that knocking down the expression of CHD1L would prevent the process of migration and invasion. So as to further verify this experimental result, cells were collected with the same treatments, and Western blotting analysis was performed to measure CHD1L protein level. E-cadherin is a transmembrane glycoprotein that mediates calcium-dependent intracellular adhesion in normal epithelial cells, while vimentin is the marker of mesenchymal cells (Wijnhoven et al. 2000). So we analyzed their levels in the negative control group and siRNA group. Then, we can see that in the siRNA4 group, the expression of epithelial markers E-cadherin and b-catenin increased, while the expression of mesenchymal markers N-cadherin and vimentin decreased (Fig. 5c). Thus, the high expression of CHD1L perhaps participated in EMT and affected certain actions of cells. In conclusion, we could clarify that
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Cell Mol Neurobiol Fig. 5 Knockdown of CHD1L will inhibit the migration of glioma cells. a Wound healing assays with control-siRNA and CHD1L-siRNA-4 cell lines. The migration of the cells to the wound was visualized at 0, 12, and 24 h with an inverted Leica phase-contrast microscope (9200 magnification). P \ 0.05. b Crystal violet staining of glioma cells that crossed the polycarbonate membrane of the trans-well chamber to detect the migration of cells. c Western blot analysis of CHD1L, E-cadherin, bcatenin, N-cadherin, vimentin, and GAPDH in control-siRNA and CHD1L-siRNA-4 cell lines (Color figure online)
CHD1L was related to the migration and invasion of glioma.
Discussion Glioma, arising from glial cells, remains one of the most aggressive primary CNS tumors (Ferguson 2011). Although diagnosis and therapy of glioma have been investigated for decades, the incidence and mortality rates still stay at a high level (Liu et al. 2010; Taylor 2010). Furthermore, glioma shows a high percentage of recurrence and migration and invasion through the activation of relevant genes, which leads to a poor prognosis (Tao et al. 2012; Taylor 2010). Thus, it is a urgent task for clinical
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practitioner and basic scientists to explore new molecular markers and relevant mechanisms. Recently, amplification of 1q21 is one of the most frequent genetic alterations in many solid tumors, including bladder, breast, nasopharyngeal carcinoma, hepatocellular carcinoma, esophageal tumor, and colorectal carcinoma (Cheng et al. 2013; He et al. 2012; Hyeon et al. 2013; Ji et al. 2013; Tian et al. 2013). CHD1L is a recently identified oncogene that is frequently amplified in hepatocellular carcinoma (HCC) (Ma et al. 2008). It belongs to the SNF2 superfamily, containing SNF2-N domain and a helicase superfamily domain. Therefore, CHD1L has also been hypothesized to play important roles in transcriptional regulation, maintenance of chromosome integrity and DNA repair (Bork and Koonin 1993; Karras et al. 2005; Ryan and Owen-Hughes 2011).
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In this study, we demonstrated that CHD1L might be an important regulator in glioma. Firstly, Western blot analysis using samples from seven glioma tissues (grade II–IV) revealed that CHD1L was overexpressed in glioma tissues and its expression was significantly higher in high-grade glioma tissues than in low-grade glioma tissues (Fig. 1a, b). Secondly, immunohistochemistry stain of 81 glioma samples showed that immunoreactivity of both CHD1L and Ki-67 was seen predominantly in the nucleus, and the positive ratio of their expression was both increased with WHO grade (Fig. 1c). In addition, immunohistochemistry analysis revealed that CHD1L expression was correlated with Ki-67. Multivariate analysis with the Cox proportional hazards model indicated that CHD1L could be an independent prognostic factor for the survival of glioma patients. Accordingly, Kaplan–Meier analysis revealed that CHD1L overexpression predicted poor survival. Functional studies of CHD1L in hepatocellular carcinoma suggested that its oncogenic role in tumorigenesis is through unleashed cell proliferation, G1/S transition, and inhibition of apoptosis (Hyeon et al. 2013). In our study, serum starvation and release experiments showed that after serum readdition, CHD1L was gradually increased in accordance with cyclin D1 and PCNA, which were considered to be related with the cell cycle of glioma. It has been reported that the p53 pathway is crucial for effective tumor suppression in humans (Green and Chipuk 2006). And the p53 protein can upregulate the expression of p21, which in turn functions as a Cdk2 inhibitor to control S phase entry via the inactivation of cyclinE–Cdk2 complex (Chen et al. 2009b; Zolota et al. 2007). Consistent with this theory, we found that loss of CHD1L resulted in concomitant increased expression of p53 and p21, while reduced expression of cyclinE and Cdk2. These evidences suggested that the dysregulation of the p53–cyclinE–Cdk2 pathway might be involved in CHD1L-induced G1/S transition in glioma. According to Chen et al. (2009a), CHD1L activation may disrupt the cell death program via binding the apoptotic protein Nur77 or through activation of the AKT pathway by up-regulation of CHD1L-mediated target genes. In our study, knockdown of CHD1L resulted in increased expression of active caspase 3 and inhibited expression of Bcl-2, which indicated that CHD1L might suppress nuclear to mitochondrial translocation and inhibit the subsequent caspases activation and cell death. Finally, with wound healing assay and trans-well assay, we found that CHD1L was related to the migration and invasion of glioma. The epithelial-mesenchymal transition (EMT) includes loss of cell–cell adhesion and activation of mesenchymal markers as well as increased motility of tumor cells, which suggests that EMT is a major mechanism of tumor migration and invasion (Friedl and Wolf
2003; Kang and Massague 2004).To better elucidate the invasive and metastatic mechanisms of CHD1L, the effect of CHD1L depletion on the EMT was investigated. As expected, the epithelial markers E-cadherin and b-catenin were downregulated, whereas the mesenchymal markers N-cadherin and vimentin were increased in CHD1Ltransfected cells. It was reported that reduced E-cadherin expression could result in adherens junction break-down, and loss of cell polarity ensues, that is accompanied by increased stability and cytoplasmic accumulation of the adherens junction component b-catenin (Singh and Settleman 2010). Taken together, the EMT induced by CHD1L is an important mechanism underlying glioma development and migration. In summary, we had shown that CHD1L overexpression occurred in glioma specimens and was associated with glioma grade as well as poor prognosis. Furthermore, knockdown of CHD1L expression by siRNA could affect the cell cycle, apoptosis, and migration of glioma. Our findings indicated that CHD1L may be a novel candidate gene for the future development of diagnosis and therapeutic strategies for glioma. Nonetheless, more mechanisms of CHD1L genomic functions in glioma and other tumors need to be investigated to pave the way for novel therapeutic strategies. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 81201858, 81272789), the National Natural Science Foundation of Jiangsu province (No. BK2012231), and the Nantong Society Undertaking and Technological Innovation (HS2014069). Compliance with Ethical Standards Conflict of interest
None.
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ORIGINAL RESEARCH
CHD1L Regulates Cell Cycle, Apoptosis, and Migration in Glioma Jie Sun1 • Li Zhang2 • Hongyu Zhao1 • Xiaojun Qiu1 • Wenjuan Chen1 • Donglin Wang2 • Na Ban2 • Shaochen Fan2 • Chaoyan Shen1 • Xiaojie Xia1 Bin Ji1 • Yuchan Wang2
•
Received: 12 May 2015 / Accepted: 6 July 2015 Ó Springer Science+Business Media New York 2015
Abstract Chromodomain helicase/ATPase DNA binding protein 1-like (CHD1L) gene is a newly identified oncogene located at Chr1q21 and it is amplified in many solid tumors. In this study, we intended to investigate the clinical significance of CHD1L expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemistry analysis showed that CHD1L was overexpressed in glioma tissues and glioma cell lines. In addition, the expression level of CHD1L was positively correlated with glioma pathological grade and Ki-67 expression. Kaplan–Meier curve indicated that high expression of CHD1L may result in poor prognosis of glioma patients. Accordingly, suppression of CHD1L in glioma cells was shown to induce cell cycle arrest and increase apoptosis. In addition, knockdown of CHD1L significantly accelerated migration and invasion ability of glioma cells. Together our findings suggest that CHD1L is involved in the progression of glioma and may be a novel target for further therapy.
Jie Sun and Li Zhang have contributed equally to this work. & Bin Ji [email protected] & Yuchan Wang [email protected] 1
Department of Radiotherapy and Oncology, The Affiliated Hospital of Nantong University, Xisi Road No. 20, Nantong 226001, People’s Republic of China
2
Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College of Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
Keywords Migration
CHD1L Glioma Cell cycle Apoptosis
Introduction Malignant glioma, highly invasive primary brain tumor, is the most frequent type of primary adult brain neoplasms (Venkataramanaa et al. 2013; Wen and Kesari 2008). WHO classification based on the malignancy of the neoplasms ranges from grade I, corresponding to low-proliferative noninvasive tumors, up to grade IV, assigned to cytologically malignant, highly infiltrative, mitotically active, and necrosis-prone glioblastoma multiforme (GBM, malignant glioma) (Sims et al. 2015). Current standard of care includes maximal safe surgical resection, fractionated radiation, and systemic temozolomide chemotherapy (Zhuang et al. 2009). Despite advances in conventional treatment, the outcome for glioma patients remains almost universally fatal. This poor prognosis is mainly due to therapeutic resistance and tumor recurrence after surgical removal (Wang et al. 2015). Therefore, it is urgent to reveal the underlying main molecular mechanisms of this malignancy so as to develop more effective therapies and improve the disappointing outcome. The chromodomain helicase DNA binding protein 1-like (CHD1L) gene is a putative oncogene mapped to chromosome 1q21 (Cheng et al. 2013). The CHD family of proteins is ATP-dependent chromatin remodeling enzymes which combine chromodomains with SWI2/SNF2 ATPase/ helicase motifs and have DNA binding capability (Cheng et al. 2013). The clinical significance of amplification and overexpression of CHD1L has been evaluated in solid tumors, including HCC (Hyeon et al. 2013), ovarian carcinoma (He et al. 2012), colorectal carcinoma (Ji et al.
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2013), and bladder cancer (Tian et al. 2013). All these studies demonstrate that CHD1L is a novel biomarker for prediction of progression, prognosis, and survival. However, to the best of our knowledge, there is as yet no report demonstrating the function and mechanism of CHD1L in glioma. In this study, we further verified the high expression of CHD1L in glioma tissues and cell lines, and then, we analyzed its relationship with clinicopathological features and its prognostic value for glioma patients’ survival. Furthermore, we investigated the possible role of CHD1L in cell cycle, apoptosis, migration, and invasion of glioma. According to our research, it was reasonable to consider that CHD1L could be a novel therapeutic target for glioma.
Materials and Methods Patients and Tissue Samples For Western analysis, all fresh frozen human glioma tissue samples were stored at -80 °C immediately after surgical removal. While for immunohistochemical analysis, a total of 81 glioma specimens were obtained from the Department of Pathology, Affiliated Hospital of Nantong University from 2005 to 2011. None of the patients were treated with such preoperative therapies as immunotherapy, radiation, or chemotherapy. All patients were followed up for 1–60 months. For histological examination, all tissues were fixed in 10 % buffered formalin and embedded in paraffin for sectioning. The glioma samples collection was executed in accordance with the protocols approved by the Ethics Committee of the Affiliated Hospital of Nantong University. The main clinical and pathologic variables are shown in Table 1. Immunohistochemical Staining Surgically excised tissues were fixed with 10 % formalin and embedded in paraffin, and 4-lm-thick specimen sections were prepared on glass slides. Tumor tissue microarray blocks were deparaffinized using a graded ethanol series, and endogenous peroxidase activity was blocked by soaking in 0.3 % hydrogen peroxide. Then, the sections were processed in 10 mmol/L citrate buffer (pH 6.0) and heated to 121 °C in an autoclave for 3 min to retrieve the antigen. Hydrogen peroxide (0.3 %) was applied to block endogenous peroxide activity for 20 min after cooling. After rinsing in PBS (pH 7.2), the sections were incubated with mouse anti-human CHD1L antibody (diluted 1:50) and mouse anti-human Ki-67 antibody (diluted 1:400) for 2.5 h at room temperature. All slides were processed using the peroxidase–antiperoxidase method
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(Dako, Hamburg, Germany). After washing with PBS, the peroxidase reaction was visualized by incubating the sections with DAB (0.1 % phosphate buffer solution, 0.02 % diaminobenzidinetetrahydrochloride, and 3 % H2O2). After being rinsed in water, the sections were counterstained with hematoxylin. At last, the sections were dehydrated with graded alcohol and cover slipped. Immunohistochemical Analysis The immunostained sections were evaluated by three pathologists without knowledge of the clinical and pathological parameters of the patients in a blinded manner. For assessment of CHD1L and Ki-67 expression, at least five high-power fields were chosen randomly in each specimen, and more than 300 cells were examined to determine the mean percentage of signal-positive cells. The staining results were scored semiquantitatively. The percentage of cells was scored as follows: 1 (0–49 % tumor cells stained), 2 (50–74 % tumor cells stained), and 3 (75–100 % tumor cells stained). The intensity of staining was coded as follows: 1 (weak staining), 2 (moderate staining), and 3 (strong staining). Then, we multiplied the two scores and classified them into two groups: low expression (0–4.5) and high expression (4.5–9). As for statistical analysis of Ki-67 stain, a cutoff value was used to distinguish tumors with a low (\50 %) or high (C50 %) level of Ki-67 expression. Antibodies The antibodies used for Western blot analysis and immunohistochemistry were as follows: anti-CHD1L monoclonal antibody (Santa Cruz Biotechnology, USA), antihuman Ki-67 monoclonal antibody (Santa Cruz Biotechnology, USA), anti-human proliferating cell nuclear antigen (PCNA) monoclonal antibody (Santa Cruz Biotechnology, USA), anti-human cyclin D1,anti-human cyclin E polyclonal antibody (Santa Cruz Biotechnology, USA), p53, p21(Santa Cruz Biotechnology, USA), E-cadherin, b-catenin, N-cadherin, vimentin(Santa Cruz Biotechnology, USA),and anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibody (Santa Cruz Biotechnology, USA). Cell Cultures The human glioma cell lines U87MG, U251MG, U118MG, A172, and H4 were obtained from the cell library of the Chinese Academy of Sciences and were cultured in Dulbecco’s modified Eagle medium (DMEM) (GibCo BRL, Grand Island, NY, USA) with 10 % fetal bovine serum, 2 mM L-glutamine, and 100 U/mL penicillin–streptomycin
Cell Mol Neurobiol Table 1 CHD1L expression and clinicopathological variables in 81 glioma specimens
Variables
Total
v2 value
CHD1L expression Low score \5
P value
High score C5
Age (years) \50
47
20
27
C50
34
18
16
0.855
0.355
0.048
0.827
4.164
0.384
1.052
0.591
0.046
0.829
12.542
0.002*
21.287
0.001*
Gender Male
48
23
25
Female
33
15
18
Tumor location Frontal
23
9
14
Parietal
10
5
5
Occipital
11
4
7
Temporal Unknown
17 20
11 12
6 8
Extent of resection Biopsy
16
9
7
Total resection
28
12
16
Subtotal resection
37
20
17
Tumor size (cm) \4
17
9
8
C4
64
32
32
II
22
18
4
III
29
13
16
IV
30
10
20
Low expression
56
28
28
High expression
44
10
34
WHO grade
Ki67
Statistical analyses were performed by the Pearson v2 test * P \ 0.05 was considered significant
mixture (GibCo BRL) at 37 °C, and 5 % CO2. The medium was changed every 2–3 days, and cultures were split using 0.25 % trypsin. Western Blot Tissue and cell protein were promptly homogenized in a homogenization buffer containing 1 M TrisHCl pH 7.5, 1 % Triton X-100, 1 % Nonidet p-40 (NP-40), 10 % sodium dodecyl sulfate (SDS), 0.5 % sodium deoxycholate, 0.5 M EDTA, leupeptin 10 lg/mL, aprotinin 10 lg/mL, and 1 mMPMSF, and then centrifuged at 10,0009g for 30 min to collect the supernatant liquid. Protein concentrations were determined with a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). The total cellular protein extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride filter (PVDF) membranes (Millipore, Bedford, MA). After the membranes
were blocked in 5 % nonfat milk in TBST (150 mMNaCl, 20 mMTris, 0.05 % Tween 20) for 2 h, they were incubated with the primary antibodies overnight at 4 °C. Then, the membranes were washed with TBST for three times, 5 min each, and then horseradish-peroxidase-linked IgG as the secondary antibodies for 2 h at room temperature. The membrane was developed using the ECL detection systems. The experiments were carried out in three separate occasions. Transient Transfection Cells were grown in dishes until they reached 80 % confluence. The medium was replaced 6 h later with fresh medium for transfection. The CHD1L-siRNA and control-siRNA were purchased from Genechem (Shanghai). The CHD1L-specific siRNA target sequence: CHD1L-siRNA#1 was 50 -GTATTGGACATGCCACGAAA-30 , CHD1L-siRNA#2 was 50 -TAT TGGACATGCCACGAAA-30 , CHD1L-siRNA#3 was 50 -
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CAAGAGAAGGAGACTCATA-30 , and CHD1L-siRNA#4 was 50 -ACAAACTCTTGCAGCCATT-30 . Glioma cells were transfected with CHD1L-siRNA or control-siRNA according to the manufacture’s instructions. Cells were collected for Western blot, CCK-8, and flow cytometry assays after transfection for 24–48 h. Flow Cytometry Analysis of Cell Cycle and Cell Apoptosis To synchronize the cell cultures, we seeded U87 cells in 6-cm dishes in growth medium with 10 % FBS overnight. Then, the cultures were rinsed by PBS and changed to serum-free medium. After serum starvation for 72 h, the cells were released into cell cycle by addition of 10 % FBS. For flow cytometrical analysis of cell cycle distribution, cells were collected and fixed in 70 % ethanol over night at 4 °C, washed with PBS, and then incubated with 1 mg/mL RNase A for 30 min at 37 °C. Then, cells were stained with 50 mg/mL propidium iodide (PI) (Becton– Dickinson, San Jose, CA) in PBS and 1 % Triton X-100. The data were acquired using a BD FACSCAN flow cytometer (Becton–Dickinson, San Jose, CA) and analyzed using the Cell Quest Acquisition and analysis programs (Becton–Dickinson). U87 cells transfected with CHD1LsiRNA and control-siRNA were cultured for 48 h and harvested. Then, add 60 lL of MuseTMAnnexin V & Dead Cell Reagent (Part No. 4700-1485, 100 tests/bottle) to each tube and add 60 lL of cells in suspension to each tube. After incubated for 20 min at room temperature in the dark, the apoptosis assay was performed by MuseTM Cell Analyser (EMD Millipore corporation) according to the manufacturer’s instructions. Cell Proliferation Assays Cell growth was measured using a commercial Cell Counting Kit (CCK)-8 (Dojindo, Kumamoto, Japan) in accordance with the manufacturer’s instructions. Briefly, cells were plated into a 96-well plate at a density of 2 9 104 cells/well and grown overnight. Cell Counting Kit-8 reagent was added to a subset of wells for 2 h incubation at 37 °C, and the absorbance at the wavelength of 490 nm was read in an automated plate reader. The experiments were repeated at least three times. Plate Colony Formation Assay NC and transfection of CHD1L siRNA U87MG cells (2000 cells/plate) were cultured in 5 mL of DMEM supplemented with 10 % FBS and 800 mg/mL G418 in a 6-cm plate. After 14 days, colonies were washed with PBS, fixed with methanol for 30 min, and stained with crystal violet for
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30 min. Clearly visible colonies ([50 mm in diameter) were counted as positive for growth. Monolayer Wound Healing Assays U87 cells were seeded on 6-well plates at a density of 4 9 105 cells/well. Thirty-six hours after transfection, the cells were scratched using a sterile 10 lL micropipette tip, and washed three times with PBS. Then, cells were then cultured in complete DMEM medium for an additional 48 h. Photographs were taken by an inverted Leica phasecontrast microscope (Leica DFC 300 FX) at 0, 12, and 24 h time points. Trans-well Migration Assay Into the trans-well chambers (Corning, 8 lm pore size) were seeded 1 9 105 cells in the upper chambers in medium, and DMEM with 10 % FBS was added to the bottom chambers. After 24 h of incubation, the filters were stained with crystal violet. The number of migrating cells in 5 fields was counted under 9200 magnification, and the means for each chamber were determined. Statistical Analysis All the data were carried out by SPSS17.0 statistical package from SPSS Inc. (Chicago, USA). The relationship between the CHD1L expression and the clinical features was analyzed by Chi-square (v2) or Fisher’s exact test. Correlation analysis was also used. For analyzing the survival data, Kaplan–Meier curves were constructed, and the log-rank test was performed. Cox’s proportional hazards regression model was used for multivariate survival analysis, and the risk ratio and its 95 % confidence interval were recorded for every marker. Student’s test was used for comparison between groups. P values \0.05 were considered to be statistically significant.
Results CHD1L Overexpression in Glioma Tissues To identify whether CHD1L associated with glioma or not, we evaluated the expression level of CHD1L in seven fresh glioma tissues from Grade II to Grade IV by Western blot. We found that CHD1L was overexpressed in glioma tissues, and its expression was significantly higher in highgrade glioma tissues than in low-grade glioma tissues (Fig. 1a, b). Then, we also analyzed CHD1L expression by immunohistochemistry for 81 glioma samples in order to demonstrate the association of CHD1L with glioma
Cell Mol Neurobiol Fig. 1 The correlation between CHD1L and glioma grade, Ki67 and glioma outcome. a Western blot analysis of expression of CHD1L in samples of glioma tissues (grades II–IV). b The bar chart demonstrates the ratio of CHD1L protein to GADPH for the above by densitometry. The data are mean ± SD of three independent experiments. c Paraffin-embedded glioma tissue sections (including grades II–IV) were stained with antiCHD1L antibodies and antiKi67 antibodies followed by counterstaining with hematoxylin (SP 9400). The paraffin-embedded tissue sections were stained with no antibodies as negative control (SP 9400). d The relationship between CHD1L and Ki67. e Kaplan–Meier survival curves for high CHD1L expression versus low CHD1L expression in 81 patients with glioma. On the basis of mean CHD1L percentages, patients were divided into high CHD1L expressers and low CHD1L expressers. Patients in the high expression of CHD1L group had a significantly shorter overall survival (P \ 0.01)
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progression. As shown in Fig. 1c, CHD1L localized in the nucleus of glioma cells, and its expression increased as the malignant degree increased, which was consistent with Ki67 expression. Ki-67 was considered to be associated with cellular growth and proliferation (Yamashita et al. 2010). Furthermore, there was a positive correlation between CHD1L and Ki-67 expression in the examined glioma tissues (Fig. 1d). Relationship Between CHD1L Expression and Glioma Patients’ Survival The immunohistochemical results of 81 glioma specimens are summarized in Table 1. In short, CHD1L expression was apparently associated with clinicopathologic grades (P = 0.002) and Ki67 expression (P = 0.001). Little obvious correlation was observed between CHD1L expression and patient’s gender, age, tumor location, extent of resection, or tumor size in 81 glioma cases. When a multivariate Cox proportional hazard model was constructed, we found that WHO grade, the high Ki67 expression, and CHD1L expression were prognostic factors of overall survival (P = 0.010, 0.001, 0.041; Table 2). Kaplan–Meier survival curves indicated that up-regulation of CHD1L was significantly associated with poor overall survival (Fig. 1e). Combined these results together, we inferred that high expression of CHD1L could be a strong determinant of poor prognosis in glioma. Expression of CHD1L Promotes G1/S Phase Transition Since CHD1L was correlated with the expression of Ki-67 in glioma specimens, we proposed that CHD1L might play a role in cell cycle progression of glioma cells. Among the glioma cell lines, CHD1L was expressed highly in U87 and
Table 2 Contribution of various potential prognostic factors to survival by Cox regression analysis on 81 glioma specimens Characteristic
Hazard ratio
95 % CI
P value
Age
0.821
0.433–1.554
0.544
Gender
1.522
0.783–2.959
0.216
Tumor location
1.187
0.952–1.479
0.127
Extent of resection
0.891
0.543–1.462
0.648
Tumor size
0.756
0.326–1.755
0.516
WHO grade
2.225
1.213–2.225
0.010*
CHD1L expression
2.151
1.033–4.478
0.041*
5.180–387.666
0.001*
Ki67 expression
44.814
Statistical analyses were performed by the Cox regression analysis CI confidence interval * P \ 0.05 was considered significant
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Fig. 2 The expression of CHD1L and cell cycle-related molecules in c glioma cells. a, b The expression of CHD1L in five glioma cell lines (H4, A172, U118, U87, and U251MG) and protein levels of CHD1L were quantified by correcting the average pixel intensity for each band with that of GAPDH as an internal control for equal loading of protein samples. The data are mean ± SD. c, e After serum starvation for 72 h followed by the addition of medium containing 10 % FBS for the indicated times, flow cytometry quantitation of cell cycle progress in U87 cells. d, f After serum starvation for 72 h, followed by the addition of medium containing 10 % FBS for the indicated times, flow cytometry quantitation of cell cycle progress in U251 cells. g, h U87 cells were serum starved for 72 h, and cell lysates were prepared at the indicated time points after serum refeeding and analyzed by Western blot using antibodies directed against CHD1L, PCNA, cyclin D1, and GAPDH (a control for protein load and integrity). The bar chart demonstrates the ratio of CHD1L, PCNA, and cyclin D1 protein to GAPDH for each time point by densitometry. Mean ± SEM of three independent experiments. *, #, ^P \ 0.05, compared with control cell serum starved for 72 h (S72 h). S serum starvation, R serum release, SEM standard error of the mean
U251 (Fig. 2a, b). So in the following experiment, we used them as the main experimental cell lines. To certify that CHD1L was involved in the cell cycle, we built a serum starvation and releasing model. The U87 and U251 cells were arrested in G1 phase after serum starvation for 72 h, and they were released into cell cycle by addition of 10 % FBS. Then, cells were harvested at 0, 4, 8, 12, and 24 h. After serum re-addition, the U87 cells were released from the G1 phase (from 73.19 to 41.33 %) and re-entered the S phase (from 7.42 to 38.03 %), and the U251 cells were released from the G1 phase (from 87.77 to 56.45 %) and re-entered the S phase (from 8.5 to 30.83 %). Flow cytometry results showed that the transition of G0/G1-S in U87 and U251 cells increased gradually (Fig. 2c–f). Next, Western blot was used to analyze the expression of CHD1L during cell progression, as well as the proliferation markers such as PCNA and cyclin D1. We collected the U87 cell protein at different time points, and as expected, we found that CHD1L was gradually increased after serum re-addition. Meanwhile, the cell proliferation markers PCNA and cyclin D1 had a similar tendency with the CHD1L protein level (Fig. 2g, h). These results affirmed that CHD1L may be involved in the proliferation of glioma in a cell-cycledependent pathway. Knockdown of the Expression of CHD1L Inhibited the Proliferation of Glioma Cells To further study the biologic effect of CHD1L on cell cycle, we transfected CHD1L siRNAs and control-siRNA into U87 cells to knockdown endogenous CHD1L. We used four siRNAs directed against CHD1L expression to examine their interference efficiency. U87MG cell lines were transfected with four siRNA duplexes for 48 h; the
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Fig. 3 Silencing of CHD1L decreases glioma cell growth. a We analyzed CHD1L expression by Western blot after transfected with CHD1L-siRNA in U87 cells, while CHD1L-siRNA4 achieved the best down-regulation effect. The data are mean ± SEM. *P \ 0.05 compared with the control. b Western blot analysis showing the effect of CHD1L knockdown on the protein expression of p53, p21, cyclin E, and Cdk2. c The bar chart demonstrates the ratio of CHD1L protein to GADPH for the above by densitometry (P \ 0.05).
d Growth curves of U87MG cells transfected with the CHD1L siRNA or control-siRNA. e Flow cytometric analysis of cell cycle distribution 48 h later following control-siRNA and CHDIL siRNA4 transfection. f CHD1L knockdown suppresses plate colony formation. Equal numbers of U87-control-siRNA cell and CHD1L-siRNAU87 cells were seeded onto 60-mm plates. The cells were fixed and stained with Giemsa after 14 days; the number of colonies in CHD1LsiRNAU87 cells was less than that in the con-siRNA cells
CHD1L-siRNA4 resulted in around 50 % decrease in the expression of CHD1L on protein levels compared to the control-siRNA (Fig. 3a, c). Then, we used the controlsiRNA and the CHD1L-siRNA4 for the further research. Finally, we further proved the role of CHD1L in glioma cell cycle. Suppression of endogenous CHD1L in U87MG cells inhibited the cell viability effectively and the cell cycle by CCK8 assay and flow cytometry, and plate colony formation assays indicated the same effect (Fig. 3d–f). Furthermore, DNA content analysis using flow cytometry showed that si-CHD1L was able to inhibit the cell cycle at the G1/S checkpoint (Fig. 3e). The percentage of cells in the S phase was significantly reduced in the si-CHD1Ltreated cells compared with the control-si-treated cells. It was reported that in a transgenic mouse model, CHD1L could facilitate DNA synthesis and G1/S transition through
the up-regulation of Cyclins (A, D1, and E), CDK2, 4, and down-regulation of Rb, p27 (Kip1), and p53 (Chen et al. 2009b; Liu et al. 2012). In our study, the CHD1L expression was decreased obviously and it was in accordance with the up-regulation of p53 and p21, down-regulation of cyclin E and Cdk2 (Fig. 3b). As a result, these results suggested that CHD1L may regulate cell cycle through p53–cyclinE–Cdk2 pathway and it would promote proliferation of glioma cells, and its knockdown could suppress the proliferation of glioma cells.
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Loss of CHD1L Expression Induced Apoptosis in Glioma Cells The decrease of apoptosis is another major mechanism of an oncogene in cancer development; we presumed CHD1L
Cell Mol Neurobiol Fig. 4 Effect of CHD1L downexpression on apoptosis. a U87 cells were treated with controlsiRNA and CHD1L-siRNA4, respectively, and then assayed by MuseTM Cell Analyser according to the manufacturer’s instructions. b Western blot was used for the analysis of antiapoptosis Bcl-2 and active caspase 3 expression in controlsiRNA and CHD1L-siRNA4transfected U87 cells
might be related to apoptosis in glioma. We next examined the effect of CHD1L on glioma cells’ survival. A flow cytometry assay was used to investigate whether CHD1L induced apoptosis in glioma cells. From the results we could conclude that, compared with control-siRNA-transfected cell, there were more apoptotic cells in the CHD1LsiRNA#4-transfected cells (Fig. 4a). To further study the mechanism of CHD1L in glioma apoptosis, Western assay was performed to detect apoptosis-related genes such as active caspase 3 and Bcl-2 (which considered of antiapoptotic) (Fan et al. 2014; Lin et al. 2004; Takano et al. 2014; Yang et al. 2014). As expected, knockdown of CHD1L resulted in increased expression of active caspase 3, with concomitant inhibited expression of Bcl-2 (Fig. 4b). All these results suggested that CHD1L expression might contribute to tumorigenesis through its antiapoptotic effect. CHD1L was Associated with the Migration of the Glioma Cell Line Functional studies in vitro and in vivo showed that CHD1L contributed to tumor cell migration and invasion by increasing cell motility and inducing filopodia formation and epithelial-mesenchymal transition (EMT) (Chen et al. 2010). So we supposed that CHD1L perhaps was related to
the migration and invasion of glioma. In the wound healing assay, we could see that the migration rate of the CHD1LsiRNA4 group was slower than the control group, which indicated that the ability of migration in the siRNA4 group was decreased by knocking down the expression of CHD1L compared to the negative group (Fig. 5a). Meanwhile, results from the trans-well assay revealed that knocking down the expression of CHD1L inhibited the cell migration to the bottom chambers compared to the control and negative groups (Fig. 5b). As a result, we supposed that knocking down the expression of CHD1L would prevent the process of migration and invasion. So as to further verify this experimental result, cells were collected with the same treatments, and Western blotting analysis was performed to measure CHD1L protein level. E-cadherin is a transmembrane glycoprotein that mediates calcium-dependent intracellular adhesion in normal epithelial cells, while vimentin is the marker of mesenchymal cells (Wijnhoven et al. 2000). So we analyzed their levels in the negative control group and siRNA group. Then, we can see that in the siRNA4 group, the expression of epithelial markers E-cadherin and b-catenin increased, while the expression of mesenchymal markers N-cadherin and vimentin decreased (Fig. 5c). Thus, the high expression of CHD1L perhaps participated in EMT and affected certain actions of cells. In conclusion, we could clarify that
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Cell Mol Neurobiol Fig. 5 Knockdown of CHD1L will inhibit the migration of glioma cells. a Wound healing assays with control-siRNA and CHD1L-siRNA-4 cell lines. The migration of the cells to the wound was visualized at 0, 12, and 24 h with an inverted Leica phase-contrast microscope (9200 magnification). P \ 0.05. b Crystal violet staining of glioma cells that crossed the polycarbonate membrane of the trans-well chamber to detect the migration of cells. c Western blot analysis of CHD1L, E-cadherin, bcatenin, N-cadherin, vimentin, and GAPDH in control-siRNA and CHD1L-siRNA-4 cell lines (Color figure online)
CHD1L was related to the migration and invasion of glioma.
Discussion Glioma, arising from glial cells, remains one of the most aggressive primary CNS tumors (Ferguson 2011). Although diagnosis and therapy of glioma have been investigated for decades, the incidence and mortality rates still stay at a high level (Liu et al. 2010; Taylor 2010). Furthermore, glioma shows a high percentage of recurrence and migration and invasion through the activation of relevant genes, which leads to a poor prognosis (Tao et al. 2012; Taylor 2010). Thus, it is a urgent task for clinical
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practitioner and basic scientists to explore new molecular markers and relevant mechanisms. Recently, amplification of 1q21 is one of the most frequent genetic alterations in many solid tumors, including bladder, breast, nasopharyngeal carcinoma, hepatocellular carcinoma, esophageal tumor, and colorectal carcinoma (Cheng et al. 2013; He et al. 2012; Hyeon et al. 2013; Ji et al. 2013; Tian et al. 2013). CHD1L is a recently identified oncogene that is frequently amplified in hepatocellular carcinoma (HCC) (Ma et al. 2008). It belongs to the SNF2 superfamily, containing SNF2-N domain and a helicase superfamily domain. Therefore, CHD1L has also been hypothesized to play important roles in transcriptional regulation, maintenance of chromosome integrity and DNA repair (Bork and Koonin 1993; Karras et al. 2005; Ryan and Owen-Hughes 2011).
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In this study, we demonstrated that CHD1L might be an important regulator in glioma. Firstly, Western blot analysis using samples from seven glioma tissues (grade II–IV) revealed that CHD1L was overexpressed in glioma tissues and its expression was significantly higher in high-grade glioma tissues than in low-grade glioma tissues (Fig. 1a, b). Secondly, immunohistochemistry stain of 81 glioma samples showed that immunoreactivity of both CHD1L and Ki-67 was seen predominantly in the nucleus, and the positive ratio of their expression was both increased with WHO grade (Fig. 1c). In addition, immunohistochemistry analysis revealed that CHD1L expression was correlated with Ki-67. Multivariate analysis with the Cox proportional hazards model indicated that CHD1L could be an independent prognostic factor for the survival of glioma patients. Accordingly, Kaplan–Meier analysis revealed that CHD1L overexpression predicted poor survival. Functional studies of CHD1L in hepatocellular carcinoma suggested that its oncogenic role in tumorigenesis is through unleashed cell proliferation, G1/S transition, and inhibition of apoptosis (Hyeon et al. 2013). In our study, serum starvation and release experiments showed that after serum readdition, CHD1L was gradually increased in accordance with cyclin D1 and PCNA, which were considered to be related with the cell cycle of glioma. It has been reported that the p53 pathway is crucial for effective tumor suppression in humans (Green and Chipuk 2006). And the p53 protein can upregulate the expression of p21, which in turn functions as a Cdk2 inhibitor to control S phase entry via the inactivation of cyclinE–Cdk2 complex (Chen et al. 2009b; Zolota et al. 2007). Consistent with this theory, we found that loss of CHD1L resulted in concomitant increased expression of p53 and p21, while reduced expression of cyclinE and Cdk2. These evidences suggested that the dysregulation of the p53–cyclinE–Cdk2 pathway might be involved in CHD1L-induced G1/S transition in glioma. According to Chen et al. (2009a), CHD1L activation may disrupt the cell death program via binding the apoptotic protein Nur77 or through activation of the AKT pathway by up-regulation of CHD1L-mediated target genes. In our study, knockdown of CHD1L resulted in increased expression of active caspase 3 and inhibited expression of Bcl-2, which indicated that CHD1L might suppress nuclear to mitochondrial translocation and inhibit the subsequent caspases activation and cell death. Finally, with wound healing assay and trans-well assay, we found that CHD1L was related to the migration and invasion of glioma. The epithelial-mesenchymal transition (EMT) includes loss of cell–cell adhesion and activation of mesenchymal markers as well as increased motility of tumor cells, which suggests that EMT is a major mechanism of tumor migration and invasion (Friedl and Wolf
2003; Kang and Massague 2004).To better elucidate the invasive and metastatic mechanisms of CHD1L, the effect of CHD1L depletion on the EMT was investigated. As expected, the epithelial markers E-cadherin and b-catenin were downregulated, whereas the mesenchymal markers N-cadherin and vimentin were increased in CHD1Ltransfected cells. It was reported that reduced E-cadherin expression could result in adherens junction break-down, and loss of cell polarity ensues, that is accompanied by increased stability and cytoplasmic accumulation of the adherens junction component b-catenin (Singh and Settleman 2010). Taken together, the EMT induced by CHD1L is an important mechanism underlying glioma development and migration. In summary, we had shown that CHD1L overexpression occurred in glioma specimens and was associated with glioma grade as well as poor prognosis. Furthermore, knockdown of CHD1L expression by siRNA could affect the cell cycle, apoptosis, and migration of glioma. Our findings indicated that CHD1L may be a novel candidate gene for the future development of diagnosis and therapeutic strategies for glioma. Nonetheless, more mechanisms of CHD1L genomic functions in glioma and other tumors need to be investigated to pave the way for novel therapeutic strategies. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 81201858, 81272789), the National Natural Science Foundation of Jiangsu province (No. BK2012231), and the Nantong Society Undertaking and Technological Innovation (HS2014069). Compliance with Ethical Standards Conflict of interest
None.
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