Metastasis-associated Gene 1 Expression In Human Medulloblastoma And The Role On The Mechanism Of Invasion And Metastasis

BACKGROUND:Medulloblastoma is the most common form of malignant brain tumor in children and young adults, originating in the cerebellum, posterior fossa or the vicinity of the fourth ventricle, is a major cause of morbidity and mortality in pediatric oncology.Medulloblastoma is characterized by local recurrence due to tumor cell migration and invasion. Medulloblastoms is mainly caused by secondary increased intracranial pressure due to blockage of the fourth ventricle, and commonly spreads along cerebral spine fluid pathways, but about 5% of medulloblastoma metastasize to a systemic location, especially to long bone. Surgery is still the main therapeutic choice for medulloblastoma patients,dependent on the stages of the tumor and aggressiveness of the tumor cells, medulloblastoma is also responsive to a variety of chemotherapies, radiation therapy, and combination therapies. However, the 5-year survival rate of medulloblastoma patients is still less than 5%, despite recent advances in radiotherapy and chemotherapies. Thereby, it is urgently needed to explore better treatment strategies to effectively control recurrent and progressive disease, and one of our approaches is to control the invasion and metastasis of medulloblastoma.Metastasis-associated protein (MTA) family is a group of ubiquitously expressed co-regulators comprising six gene products including metastasis-associated gene 1 (MTA1), MTAls, MTA1-ZG29p, MTA2, MTA3, and MTA3L that originate from 3 different genes. MTA1 has emerged as one of the highly deregulated oncogenes in human cancer, and its elevated levels correlate well with tumor aggressiveness and unfavorable outcomes for cancer patients in general. In the past decades, MTA1 is confirmed to have vital roles in stimulating or suppressing transcription, controlling protein steady state through influencing protein ubiquitination,leading to the DNA damage response, and keeping an overexpressed state in various human hematologic and epithelial malignancies. In addition, the critical role of MTA1 is highlighted in the nucleosome remodeling and histone deacetylase (NuRD) complex governing oepithelial-mesenchymal transition (EMT), DNA damage response, ncogenesis, and inflammation in a transcription-dependent or -independent manner. Sen at el. has reported that MTA1 protein is possibly related to the carcinogenesis and progression of different malignant cancers, which suggested MTA1 protein might be an important new tool for clinical applications in the diagnosis and treatment of various cancers.In our present study, we investigated the expression of MTA1 in human medulloblastoma by immunohistochemistry in vivo, and the significance of MTA1 expression in the invasion and metastasis of medulloblastoma by several in vitro experiments including cell transfection, reverse transcription-polymerase chain reaction (RT-PCR), western blot assay, adhesion assay, scratch assay and transwell chamber invasion assay, and further evaluated their underlying mechanisms.OBJECT:This study aims to investigate the expression of metastasis-associated gene 1 (MTA1) in human medulloblastoma, and the significance of MTAl expression in the invasion and metastasis in medulloblastoma cell line.METHODS:Study subjectsBetween December 2010 and December 2014,29 patients with pathologically confirmed medulloblastomawho underwent surgical treatment were selected from the Department of Neurosurgery in Zhujiang Hospital of Southern Medical University and Dongguan People’s Hospital. There were 15 males (51.7%) and 14 females (48.3%) aging from 5 to 36 years (median age,14.17±8.17years). Paraffin-embedded specimen tissues were reserved. The inclusion criteria were:(1) medical records of patients were complete; (2) pathologic sampling was ample and all samples were confirmed to be medulloblastoma by the Department of Pathology; (3) total or partial resection was achieved in surgery. The exclusion criteria were:(1) patients with incomplete clinical data; (2) patients with medulloblastoma accompanied by other primary tumors or with history of other primary tumors. Medulloblastoma Daoy cell line was purchased from American Tumor Cell Collection (ATCC). This study received the approval from the Ethics Committee of Zhujiang Hospital of Southern Medical University and Dongguan People’s Hospital and obtained informed consent from all patients.ImmunohistochemistryStreptavidin-perosidase (SP) immunohistochemistry was performed according to manufacturer’s protocol. Tissues from the tumor resection were fixed by 10% neutral-buffered formalin, paraffin-embedded, sliced, dewaxed and hydrated routinely. Sodium citrate buffer (pH=6.0) was applied for microwave antigen retrieval, and 0.3% H2O2 was used to block endogenous peroxidase activity for 10 min. Antibodies used were an anti-human MTA1 mousepolyclonal antibody (Abcam) at a 1:200 dilution, incubating in a 4 ℃ incubator overnight. At day 2, the tissue specimens were recovered at room temperature for 30 min, washed by phosphate buffered saline (PBS), and added with rabbit anti-rat secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Beijing, China) at 37 ℃ for 30 min. After washed with PBS, the tissue specimens were stained with diaminobenzidine (DAB, Beijing Zhongshan Jinqiao Biotechnology Co., Beijing, China), conterstained and finally mounted. PBS was utilized as a negative control.Tumor cells with pale brown or sepia particles were determined as positive cells by using a semi-quantitative couple scoring method. The staining intensity was scored as 0 for no intensity,1 for low intensity (light yellow), and 2 for moderate/high intensity (pale brown/sepia). The percentage of positive cells was then scored as:0 for unspecific staining of positive cells,1 for the percentage of stained positive cells< 20%,2 for the percentage of stained positive cells between 20% and 50%,3 for the percentage of stained positive cells> 50%. The staining results were divided into 2 grades (score of staining intensity+score of percentage of positive cells):(1)< 2, (-); (2)≥ 2, (+), immune response.In vitro experimentsSiRNA interfering MTA1 expression in Daoy cellsMTA1-targeted small interfering RNA(siRNA, target sequence: 5’-GAACATCTACGACATCTCC-3’) and nonsense control sequence niRNA were chemically synthesized.Daoy cells were transfected with MTA1-targeted siRNA (MTA1-siRNA group), niRNA (MTA1-niRNA group) and plasmid vector Lipofectamine 2000 (Invitrogen) (control group), respectively.Daoy cells were cultured in RPM-1640 culture medium in a 37 ℃,5% CO2 incubator (SANYO) to logarithmic growth phase. Lipofectamine 2000 (Invitrogen; Cytofectin, Gene Therapy Systems) was applied for the transfection of Daoy cells with MTA1-siRNA and MTA1-niRNA. Daoy cells transfected with plasmid vector were utilized as a negative control. All steps were performed according to the manufacturer’s instructions. Transfection results were observed 48～96 h after the transfection.RT-PCRTotal RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA). The purity and concentration of the isolated RNA was detected byultraviolet spectrophotometer (Analytik Jena AG, Germany). Total RNA (2μg) was reverse transcribed by using avian myeloblastosis virus (AMV) and PCR amplification was performed using complementary DNA (cDNA,5 μL). PCR reaction conditions were: denaturation at 94 ℃ for 30 s, anneal at 55 ℃ for 40 s and extension at 72 ℃ for 30 s. The primers of PCR were synthesized by Shanghai Sangon Biotechnology Co. (Shanghai, China). The upstream primer of MTA1 was 5’-CCGGGCCTGCGAGAGCTGTTACAC-3’, the downstream primer of MTA1 was 5’-CACGGCTTCCAGCGGCTTGCGTAC-3’, and the size of amplified fragments of MTA1 was 442 bp. The upstream primer of β-actin (internal inference) was 5’-GGCTACAGCTTCACCACCAC-3’,the downstream primer of β-actin was 5’-AGGAAGGAAGGCTGGAAGAG-3’and the size of amplified fragments of β-actin was 210 bp. The amplified fragments were analyzed and recorded after gel electrophoresis. The expression level of MTA1 mRNA was determined by the absorbance ratio of MTA1 amplified band to β-actin amplified band.Western blot assayDaoy cells were collected from each group and cell protein samples were extracted. The extracted samples were electrophoreticly separated by using 12% polyacrylamide gel. After trarsmembrane, the samples were sealed at room temperature, and goat anti-human MTA1 polyclonal primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a 1:200 dilution, mouse anti-human β-actin monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a 1:1000 dilution and the relevant secondary antibody were added. The samples were incubated, exposed at dark, developed, fixed and finally analyzed.Adhesion assayForty-eight hours after transfection, Daoy cellsin good condition were digested by trypsin, and seeded into 96-well plates coated with 100 μg/mL Matrigel (Becton, Dickinson and Company, NJ, USA), with a cell density of 1 x 105Daoy cells/well. Three parallel wells were set. At 30 min,60 min and 90 min after incubation, the cells were washed by PBS to remove floating cells, separately. Methylthiazolythiazolydiphenyl-tetrazolium (MTT) assay was used to determine the absorbance at 570 nm (A). The percentage of cells adhering was calculated as follows: adhesion rate (%)= A of cells adhering to Matrigel/A of total cells x 100%.Scratch assayForty-eight hours after transfection, Daoy cells in good condition were seeded into 6-well plates. The Daoy cells were scratched softly in the bottom of the 6-well plates on rectilinear path with a sterile 200 μL pipette tip after full fusion, washed with PBS twice and continued to be cultured. After 8 h,16 h and 24 h, scratch wound healing was observed under an inverted microscope separately and photographed. Cell mobility was determined by the percentage of the cells with scratch wound healing. At 0 h, the percentage of the cells with scratch wound healing was 0%.Transwell chamber invasion assayTranswell chamber (Corning, New York, USA) was put into 24-well plates, with 500 μL Dulbecco’s modified Eagle’s medium (DMEM) containing 10% calf serum added into each lower chamber, and 200 μL cell suspension into each upper chamber. Five wells were set for each group. Twelve hours after routine culture, the transwell chamber was taken out, and fixed with 95% ethanol. Then, the matrices on the inner surface of the chamber were removed by cotton swabs, followed by 4% of paraformaldehyde fixation. After stained by hematoxylin-eosin (HE), the cells was observed under a microscope and photographed.Statistical analysisThe SPSS 19.0 software was applied for statistical analysis. All measurement datawere presented as mean ± standard difference (SD). The means were compared between the two groups by using the t test; the comparisons among multiple groups were conducted by using the One-Way analysis of variance (ANOVA). Enumeration data were presented as percentage or rate, and compared by the chi-square test. All data were verified by the two-sided test. P< 0.05 was considered as statistically significant.RESULTS:1.The positive expression rate of MTA1 protein in the medulloblastoma tissues was higher than that in the adjacent normal tissues (69.0% vs.10.3%, P< 0.001);2. Statistical analysis demonstrated no statistical difference in the expression of MTA1 mRNA between the control group and the MTA1-niRNA group (P> 0.05),and the expression level of MTA1 mRNA in the MTA1-siRNA group was apparently lower than that in the control group and MTA1-niRNA group (both P< 0.05);3.As compared with the control group and MTA1-niRNA group, MTA1 protein expression level in the MTA1-siRNA group was obviously lower (both P< 0.05).,and no statistical difference in MTA1 protein expression was detected between the MTA1-niRNA group and the control group (P> 0.05);4.The results of in vitro cell adhesion analysis revealed that the adhesion rate of Daoy cells in the MTA1-siRNA group,as compared with the MTA1-niRNA group and control group (all P< 0.05),and no statistical difference was observed in the adhesion rate of Daoy cells between the control group and the MTA1-niRNA group, respectively (all P> 0.05);5.As compared with the control group and MTA1-niRNA group,the MTAl-niRNA group and control group, while scratch wound healing in the MTAl-siRNA group was relative slow, and could not reach complete healing.6.In the MTA1-siRNA group, the cell population that migrated through the microfiltration membrane apparently decreased as compared with the MTAl-niRNA group and control group (P< 0.05). No obvious difference in the cell population that migrated through the microfiltration membrane was found between the control group and the MTAl-niRNA group (P> 0.05).Conclusion:MTA1 expression increased in medulloblastoma, while MTA1 knockdown in medulloblastoma cells inhibited MTA1 expression. In addition, MTA1 knockdown inhibited the adhesion, migration and invasive abilities of medulloblastoma cells. MTA1 expression may serve as a biomarker and a potential therapeutic target for medulloblastoma.