Silencing of Forkhead Box D1 Inhibits Proliferation and Migration in Glioma Cells
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1196 ONCOLOGY REPORTS 37: 1196-1202, 2017 Silencing of Forkhead box D1 inhibits proliferation and migration in glioma cells YUAN-FENG GAO1,2, TAO ZHU1,2, XIAO-YUAN MAO1,2, CHEN-XUE MAO1,2, LING LI1,2, JI-YE YIN1,2, HONG-HAO ZHOU1,2 and ZHAO-QIAN LIU1,2 1Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, Hunan 410008; 2Institute of Clinical Pharmacology and Hunan Key Laboratory of Pharmacogenetics, Central South University, Changsha, Hunan 410078, P.R. China Received August 2, 2016; Accepted December 27, 2016 DOI: 10.3892/or.2017.5344 Abstract. Despite the extensive role of Forkhead box tran- glioma (7-10). One promising family for gene targeting is the scription factors in the development and progression of various Forkhead-box (FOX) family. The FOX family consists of at cancers, little is known about their role in glioma. We examined least 43 transcription factors that regulate the expression of the expression and function of Forkhead box D1 (FOXD1) in genes that support cell growth, proliferation and differentiation glioma cell behavior and found that FOXD1 was upregulated (11). Many FOX genes function as oncogenes. For example, and directly correlated with the glioma grade. Data analysis FOXA1 is amplified and overexpressed in esophageal and also revealed significant differences in FOXD1 expression for lung adenocarcinomas (12). FOXO1 fused to PAX3 or PAX7 both gene expression profiles (GSE4290 and GSE7696) and the serve as prognostic indicators in alveolar rhabdomyosarcoma TCGA datasets. Additionally, decreased FOXD1 expression (13-15). in U251 and U87 glioma cells caused a delay in cell growth FOXD1 (also known as FREAC-4) is highly expressed in and a disruption in colony formation. FOXD1 silencing also the kidney and controls cellularity in the renal capsule, a struc- promoted generation of apoptotic bodies containing nuclear ture required for normal renal development (16-19). FOXD1 fragments. Cells with suppressed expression of FOXD1 mark- is also expressed in the brain and the retina and is neces- edly reduced glioma cell migration. Our results suggest that sary for normal development of the retina and optic chiasm FOXD1 may serve as a novel regulator of glioblastoma cell (20,21). FOXD1 has also been implicated as an oncogene, as behavior that may offer a novel target for gene targeted glioma it promotes breast cancer proliferation and chemotherapeutic therapies. drug resistance by targeting p27 (22). However, the role of FOXD1 in tumorigenesis and progression, particularly in Introduction glioma, is still limited. In the present study, we first revealed the role of FOXD1 Gliomas are the most malignant and common type of brain in glioma tumorigenesis and progression. We found that tumors (1). Despite considerable advances in surgery, radia- FOXD1 expression is upregulated and directly correlated with tion, and chemotherapy (temozolomide, TMZ), the prognosis the glioma grade. Using FOXD1-siRNA, we determined that for glioma has not significantly improved (2). With a median silencing FOXD1 expression inhibits cell proliferation and survival time of 12-15 months, fewer than 3% of glioma migration. Our results suggest that FOXD1 may be a potential patients live longer than 5 years after diagnosis (3-5). Because therapy target for patients with glioma. of the restricted anatomical location and absence of metas- tases outside the central nervous system (CNS), targeted Materials and methods gene therapy may provide a promising strategy to treat gliomas (6). Therefore, identification of target genes that drive Cell culture and transfection. Human glioma cell lines U87 tumorigenicity is essential for developing targeted therapy for and U251 were cultured in DMEM (C11995500BT, Gibco) supplemented with 10% fetal bovine serum (FBS, 10099-141, Gibco). Cells were cultured at 37˚C in a humidified atmosphere of 5% CO2. Trypsin-EDTA (0.25%) (25200072, Gibco) was used to detach the cells from the flask. The FOXD1-siRNA Correspondence to: Professor Zhao-Qian Liu, Department of and negative control siRNA (NC-siRNA) were purchased from Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China Santa-Cruz Biotechnology (sc-60649; sc-637007) and Sigma. ® E-mail: [email protected] Cells were transfected using Lipofectamine RNAiMAX reagent (13778150, Invitrogen) according to the protocol. Key words: glioma, FOXD1, proliferation, migration, prognostic value Glioma tumor specimens. All patients undergoing surgical treatment at the Hunan Cancer Hospital (Changsha, Hunan, GAO et al: Forkhead Box D1 1197 China) for primary brain cancers between 2007 and 2013 scratched using sterile pipette tips and washed with phosphate- were invited to participate in this institutional review board- buffered saline (PBS) 3 times to remove detached cells. The approved study. Seventy patients diagnosed with low grade cells were then transfected with siRNA for 36 h. Photographs glioma (LGG, n=36) or high grade glioma (HGG, n=34) tumors of the wounded areas were obtained using a Leica DMI3000 B were included in the study. Tissue samples were collected from inverted microscope (Leica, German). The migration rate was glioma patient tumors. Normal brain tissue (n=20) was also calculated as the ratio of the width of the scratch that remained obtained from patient with brain injury. The tissue samples cell-free after migration to the initial average width of the were flash frozen in liquid nitrogen immediately after resec- scratch. The size of the wounded areas was quantified using tion and stored at -80˚C for future processing. ImageJ Version 1.41o software (National Institutes of Health). RNA extraction and quantitative real-time PCR. Total RNA Immunofluorescent staining. Glioma cells were seeded into was extracted by trizol reagent according to the manufacturer's 6-well plates and cultured with siRNA for 48 h. The cells protocol. Two micrograms of RNA was reverse-transcribed were then fixed with 4% paraformaldehyde for 10 min at room into cDNAs using the Primescript RT reagent kit with gDNA temperature, washed and stained with Hoechst 33258 (C0003, Eraser (Takara Bio Inc., Japan). The PCR system was Beyotime) for 5 min. Cells were observed under a Leica performed using SYBR Premix DimerEraser kit (Takara DMI3000 B inverted microscope equipped with ebq100-04 Bio Inc). The reactions were cycled 40 times [95˚C, 30 sec, (Leica, Germany). (95˚C, 5 sec; 55˚C, 30 sec; and 72˚C, 30 sec)] with fluorescence measurements. A melting curve was performed at the end of Statistical analysis. SPSS16.0 software was used for general amplification cycles to verify the specificity of the PCR statistics analyses. Comparisons between two experimental products. Primers used for real-time PCR were as follows: groups were performed using Student's t-test. Survival rate FOXD1, F: AAGAACCCGCTGGTGAAG; R: GTCCAGTAG was calculated using the Kaplan-Meier method with the log- TTGCCCTTGC. GAPDH, F: GAGTCAACGGATTTGGT rank test applied for comparison. All tests performed were two CGT; R: TTGATTTTGGAGGGATCTCG. All of the determi- sided and the criterion for statistical significance was p<0.05. nations were performed in duplicate. The relative expression of FOXD1 mRNA was normalized to the expression level of Results GAPDH mRNA using the 2-∆Ct method. High FOXD1 expression in glioma. The expression pattern Western blotting. Total cellular protein was extracted with RIPA. of FOXD1 was evaluated in 70 glioma tissues and 20 normal SDS-PAGE was performed with 50 µg total cellular proteins brain tissues using quantitative real-time PCR (Q-PCR). using 10% gradient Tris-glycine gels. Primary antibodies FOXD1 expres sion was significantly higher in glioma tissues used included anti-FOXD1 (WH0002297M1, Sigma-Aldrich (LGG, p=0.047; HGG, p=0.002) compared with normal brain LLC, USA), and anti-GAPDH (G8795, Sigma-Aldrich LLC). samples. FOXD1 expression was also higher in high grade The bound antibodies were visualized using an enhanced glioma compared with low grade glioma (p=0.001, Fig. 1A). chemiluminescence reagent (RPN2232, GE Healthcare, UK) Furthermore, the asso ciation between FOXD1 expression and and quantified by densitometry using ChemiDoc XRS+ image clinicopathological parameters was analyzed in the present analyzer (Bio-Rad, USA). Densitometric analyses of bands study. No significant association was observed between were adjusted with GAPDH as loading control. Triplicate FOXD1 expression and patient age or gender (Table I). To experiments with triplicate samples were performed. further validate these results, we analyzed expression of FOXD1 in expression profiles (GSE4290 and GSE7696) MTS assay. Cells were collected in the logarithmic phase of acquired from the Gene Expression Omnibus (GEO, http:// growth and seeded into 96-well culture plates (1,000 cells/well). www.ncbi.nlm.nih.gov/geo/) database, and glioma datasets The cells were incubated in 100 µl MTS reagent (1:9) for acquired from The Cancer Genome Atlas (TCGA; http:// 1 h. The absorbance values were measured at 490 nm by the cancergenome.nih.gov/). We found that expression of BioTek ®Eon (Synergy™, HT, USA). In the absence of cells, upregulated FOXD1 (>2-fold upregulated) expression was background absorbance of the medium was subtracted. Each significantly higher in malignant gliomas compared to lower assay was performed in triplicate. grade gliomas and non-tumor brain tissue and directly corre- lated with the glioma grade in GSE4290 dataset (Fig. 1B). Colony formation assay. U251 cells were collected in the loga- Analysis of the GSE7696 data and TCGA revealed that rithmic phase of growth and seeded in triplicate into 6-well FOXD1 have >2-fold upregulation at the transcription level plates at a density of 1,000 cells/well. Cells were cultured for and were greatly increased in malignant gliomas when 12 days at 37˚C in an incubator with a 5% CO2 atmosphere. compared to non-tumor brain tissue (Fig. 1C and D). These The cells were then fixed with 4% paraformaldehyde for results suggest that increased FOXD1 expression correlates 30 min and stained with Giemsa (C0121, Beyotime, China) with glioma tumor grade.