Silencing Kif2a Inhibits Growth And Metastasis In Squamous Cells Carcinoma Of The Oral Tongue

Background:Oral cavity cancer consistently ranks as one of the ten most frequently diagnosed cancers in the world, especially, squamous cells carcinoma of the oral tongue (SCCOT) is one of the most common tumors of the head and neck. Recurrence and metastasis is the main cause for the Patients' poor prognosis.Studies have shown that cytoskeletal reorganizations play an important role in the migration of neoplastic cells. As a fundamental cytoskeleton, microtubules (MTs) are not only essential for mitotic activity of malignant cells but also for invading neighboring tissues and for the distant metastasis. The decrease and depolymerization of MTs are associated with the metastatic potentiality of the malignant tumors.Members of Kinesin-13 family including Kif2a, Kif2b and Kif2c are classified a MT depolymerase that depolymerize MTs at their ends. Among them, Kif2a specifically localizes to centrosomes during mitosis. Either depletion of Kif2a by siRNA in human tumor cells or addition of Kif2a antibody to the Xenopus eggs leads to the formation of monopolar spindles and a block in cell cycle progression. These findings suggest that Kif2a is necessary for bipolar spindle assembly and normal mitosis progression.Kif2a is also necessary in non-mitotic cells. Previous studies showed Kif2a was required for Proper neuronal outgrowth and its depletion could cause neuronal migratory delay by changing MTs dynamics around the cell bodyBased on mentioned above, we speculate that Kif2a may be involved in changing cytoskeleton and mitosis of the cells by regulating the MT dynamics. However, up to now, very few study on the relationship between Kif2a and malignant tumors, especial SCCOT, has been reported. Our study aimed to demonstrate the Kif2a expression and its potential roles in SCCOT cell progression, invasion and metastasis. And we also investigated the effect of Kif2a gene-silence on Tca8113 cells. Finally, Genechip analysis was performed to select abnormal expression genes and to investigate the molecular mechanism of Kif2a regulate proliferation, apoptosis, invasion and migration of cells, and to investigate the significance of gene silencing targeting Kif2a in the treatment of squamous cell carcinoma of the oral tongue.Expression and significance of Kif2a in squamous cells carcinoma of the oral tongueObjective:To study the expression of Kif2a in squamous cell carcinoma of the oral tongue and its correlation with various clinicopathologic parameters, then to investigate the effect of Kif2a overexpression on tumor proliferation, invasion and metastasis.Meterial and Methods:1. Tissue samples:Tissue samples from surgically resected tongue squamous cell carcinomas and their corresponding clinical information were obtained from Stomatology Hospital of Shandong University between 2005 and 2008 after obtaining written informed consent from 44 patients. And all these 44 patients underwent neck dissection,20 of them have lymph node metastasis. All the diagnoses were made following the Pathology and Genetics of Head and Neck Tumors of World Health Organization Classification of Tumors. 2. Immunohistochemistry (IHC) staining:The score of Kif2a expression was assessed based on the immunohistochemical examination. The correlation between the expression of Kif2a and various clinicopathologic parameters was investigated.Results:1. Immunohistochemistry showed that 31 of 44 cases of SCCOT (70.5%) overexpressed Kif2a compared with the paired adjacent tissues. The strong positive staining is seen in the cytoplasm and neucleus of cancer cells and the weak staining is observed in that of the adjacent epithelium (5.09±1.217 vs.4.20±1.133), which was significantly different (P=0.002).We also investigated the Kif2a expression in the primary SCCOT and the paired lymph nodes containing metastasizing carcinoma cells by immunohistochemistry.14 of 20 cases (70%) showed stronger Kif2a expression in the metastatic carcinoma cells in the lymph node than in the paired primary SCCOT (6.60±0.754 vs.5.50±0.889) (P=0.001).In addition, stronger Kif2a expression was seen in clinical Stage III/IV tumors (not shown) than clinical StageⅠ/Ⅱtumors(not shown) (P=0.001), and the primary tumors with lymph node metastasis also showed stronger Kif2a staining (not shown) than those without lymph node metastasis (not shown) (P=0.038). There were no significant differences between sex, age, tumor grade, and histological classificationConclusion:Kif2a was overexpressed in SCCOT compared with the paired adjacent tissues, stronger Kif2a expression also can be seen in the metastatic carcinoma cells in the lymph node than in the paired primary SCCOT. Its expression was associated with lymphatic metastasis and cilinical stage. Kif2a might be a novel prognostic factor for SCCOT.The effect of gene-silence Kif2a on the proliferation, apoptosis, invasion and migration of the human tongue squamous cell carcinoma cells Tca8113 cells Objective:1.To investigate the effect of Kif2a gene-silence on cell growth, apoptosis, and invasion in Tca8113 cells.2. To investigate the effect of both 5-Fu and Kif2a gene-silence on growth of human squamous cell carcinoma of the oral tongue.2. To investigate the effect of Kif2a gene-silence on growth of human squamous cell carcinoma of the oral tongue in BALB/c nude mice model.Meterial and Methods:1.Cell culture:Human tongue squamous cell carcinoma cell lines (Tca8113 cells) were routinely cultured in DMEM containing 10% foetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100μg/ml) at 37℃in a humidified air atmosphere containing 5% carbon dioxide.2. pGPU6/GFP/Neo-Kif2a and pGPU6/GFP/Neo-NC were established and evaluated by Shanghai Genepharma Co., Ltd.3. Gene silencing with siRNA:Tca8113 cells, divided into three groups for transfection:lipofectamine 2000 only (mock), pGPU6/GFP/Neo-NC, or pGPU6/GFP/Neo-Kif2a, were plated at a concentration of 2×105 cells per well in six-well plates. Growing to 70% confluence, the cells were transfected with the pGPU6/GFP/Neo-Kif2a premixed with the Lipofectamine 2000 (Invitrogen, USA) in Opti-MEM (Invitrogen, USA). pGPU6/GFP/Neo-NC was used as a negative control. The sequence of Kif2a-siRNA (Ambion,USA) was: '5-GGCAAAGAGAUUGACCUGG-3'.Semi-quantitative reverse transcription polymerse chain reaction (RT-PCR) analysis and Western blot analysis for Kif2a. Total RNA was prepared using Trizaol reagent (Invitrogen, USA) according to the manufacturer's protocol, and cDNA was synthesized by the First Strand cDNA Synthesis kit (Fermentas, Lithuania). The primers were synthesized by the Shanghai Sangon Biological Engineering Technology & Services Co.Kif2a expression was measured on protein level by Western blot.4. Stable cell lines generation:Tca8113 cells stably expressing pGPU6/GFP/Neo-Kif2a or pGPU6/GFP/Neo-NC were generated using G418-selection methods. Finally, the Tca8113 cells stably expressing pGPU6/GFP/Neo-Kif2a (Tca8113-Kif2a) or pGPU6/GFP/Neo-NC (Tca8113-NC) were grown up and lysed for Western blot assays.5. Cell proliferation assay:Cell growth of three groups Tca8113 cells were tested by MTT and Soft-agar colony-formation assay after silencing gene of Kif2a. The cells were incubated overnight at a density of 5,000 cells/well in 96-well plates. Then the growth curve was draw after 1,3,5,7,9 days to assess Tca8113 cell proliferation activity.Or after plated 24h, the cells were treated with 5-Fu of different concentration and then incubated for 4 days.20μl of MTT was added each well and color development was measured on a microplate reader at 490 nm.After 14 days culture in soft agar, cells were stained with 0.05% crystal violet. Cell colonies were counted using Image-proPlus 6.0 software from Media Cybernatics, Inc. (Bethesda, USA).6. Cell apoptosis analysis:Tca8113 cells of three groups were plated at a concentration of 2×105 cells per well in six-well plates. Growing to 80% confluence, the cells were collected and labeled with annexin V-biotin followed by PI. Annexin V-PI were measured by FACS Calibur and analyzed with the Modfit Software.7. Cell migration assay:The Tca8113 cells were added into the transwell inserts and were incubated for 24 hours. After incubation, the invaded cells were counted in five fields for each filter under a light microscope at 400x magnification.8. Establishment of nude mice tumor model. Male BALB/c nude mice of four weeks old was injected submucously with 0.1ml Tca8113 cells (cell number:2×105) under the anesthesia with aether. The nude mice were breed under SPF condition. The diameter of the neoplasma was measured every four days and the volume was calculated.35 days later, the nude mice were executed and the tumors were weighed.Results:1.The mRNA expression of Kif2a decreased by 90% 48h later and the protein expression decreased significantly 72h later after transfected by pGPU6/GFP/Neo-Kif2a (P=0.024).2. Western blot assay showed that expression of Kif2a is significantly decreased in Tca8113-Kif2a cells by 76.5%. On the contrary, there is no alteration of Kif2a expression in Tca8113-NC cells and maternal Tca8113 cells.3. MTT results showed Tca8113-Kif2a cells grew more slowly than Tca8113-NC and Tca8113 cells (after 3,5,7,9 days Tca8113-Kif2avs Tca8113-NC:P=0.013,0.015, 0.017,0.005); while there was no significant difference between the Tca8113-NC and Tca8113 cells.4. MTT results also showed the IC50 of 5-Fu in Tca8113-Kif2a cells decreased significantly than the other groups of Tca8113 cells (Tca8113-NC vs Tca8113-Kif2a: IC50=32.55±2.5430vs14.745±2.3600, P=0.024).5. Cell colonies showed that significant less cell colonies in Tca8113-Kif2a than Tca8113-NC and Tca8113 cells (281±17 vs 143±12, P<0.01).6. Apoptosis is induced in Tca8113-Kif2a cells:The percentage of apoptotic cells of Tca8113-Kif2a significant increases as compared to Tca8113-NC cells ((28.94±3.739)%vs (6.617±1.263)%; P=0.014).7. Transwell analysis showed the invasiveness of Tca8113-Kif2a cells decreased dramatically in comparison to the Tca8113-NC cells (1386±184.45 vs 646±55.00, P=0.014).8. The hydropsia formed first after injecting Tca8113 and Tca8113-NC cells. A node could be touched four days later. The tumor grew continuously, round or ellipse shaped. Ten days later a neoplasma formed in a diameter about 3～5mm. On the contrary the neoplasma of nude injected by Tca8113-Kif2a cells grew slowly and the volume was smaller(1505±391.5 vs 2816±396.3 mm3,P<0.05); the weight was slighter than injected by Tca8113-NC cells(1.085±0.149 vs 2.979±0.532g, P<0.05).Conclusion:1. pGPU6/GFP/Neo-Kif2a could inhibit the expression of Kif2a gene and protein in Tca8113 cells. The proliferation activity and invasion decreased in Tca811-Kif2a cells, and Kif2a gene-silence could inhibit the proliferation of nude tumor, which meant inactivation of Kif2a gene might be a promising strategy for SCCOT. 2.Kif2a gene-silence could increase inhibition rates of 5-Fu on the proliferation of Tca8113 cells of tongue squamous cell carcinoma.3. Kif2a was an ideal target gene for gene therapy of tumor proliferation of SCCOT.Influence of of Kif2a gene-silence on the gene expression profile of the human tongue squamous cell carcinoma cells Tca8113Objective:1. To analysis the influence of Kif2a gene-silence on human gene expression profile of Tca8113 cells, to select abnormal expression genes from the gene chips and to investigate the molecular mechanism of Kif2a regulate proliferation, apoptosis, invasion and migration of Tca8113 cells.2. To investigate the significance of gene silence targeting Kif2a in the treatment of squamous cell carcinoma of the oral tongue.Meterial and Methods:1.Genechip hybridization and data analysis of gene-silence of Kif2a on Tca8113 cells.5×106 cells of every group were collected and extracted total RNA by Trizol.Then Genechip hybridization and data analysis were performed by KangChen Bio-tech, Shanghai, China.2. Some differentially expressed genes were verified with real time RT-PCR.Results:1. Analysis of gene expression profile showed there were 2093 genes expression have significant difference between Tca8113-Kif2a and Tca8113-NC cells,which including 48 uP-regulated genes and 18 down-regulated genes in Tca8113-Kif2a cells. These genes are related to many important singal pathways such as cell cycle/mitosis, apoptosis and PI3K/Akt signal pathways.2. Some differentially expressed genes (PI3K%,Akt%,Bcl-2, Bax and MMP-9)were verified with real time RT-PCR.The Ratio is similar to that of Genechip analysis. Conclusion:1. Kif2a gene silence could lead to change of some genes of human gene expression profile,which are assoiated with some important signal pathways.2. Kif2a may regulate the proliferation,apoptosis,invasion and migration of Tca8113 cells through PI3K/Akt singal pathway.