Effects Of DEK Gene On Biological Behaviors And Clinical Significance In Colorectal Carcinoma

Backgrounds:Colorectal carcinoma (CRC) remains the most common human malignant tumor in the worldwide and it still is one of the leading causes of cancer-related death. The postoperative5-year survival rate of CRC is only about65%. Targeted therapies to cancer may be more effective and less harmful to normal cells than current treatments. The next stage of targeted therapies will focus on finding which gene silencing will respond to therapies. DEK, emerging as a novel class of DNA topology modulators which can be both targets and effectors of protumorigenic events, located at chromosome6p22.3, a highly conservative nucleoprotein can being phosphorylated, and composed of375amino acids mainly distributed in nucleus euchromatin, and preferentially expressed in actively proliferating and malignant cells, where it can reach up to4to6million copies per nucleus. Recently, DEK was reported as a gene which was frequently up-regulated in aggressive human tumors. However, to date, the expression status of DEK in colorectal cancer, its relationship with clinic-pathological features/prognosis, and its effect on biological behaviors of colorectal cancer cell are unknown.Objectives:To detecte the effects of DEK gene on biological behaviors in colorectal carcinoma and explore the molecular mechanism related by cytological experiments, and evaluate the clinical significance of DEK gene for colorectal carcinoma prognosis by histological experiments.Materials and methods:1. Cytological experiments:The levels of DEK mRNA and protein expression in colon cancer cell HCT116, HT29, SW480, and SW620were detected by RT-PCR and Western blot. Immunofluorescence staining method and Confocal Laser Scanning Microscope were used to determine the DEK protein localization in SW620cell. After knockingdown the DEK expression, detection the cell proliferation was using by MTT assay and colony formation assay, and Hoechst33342staining and Flow cytometry were used to detect the cell apoptosis level of SW620cells. The motility capacity of SW620cell transfected by siDEK was showed by wound healing assays. Moreover, we uses Western blot to detected the proliferation, apoptosis and migration related factors levels, such as mutant-p53and MDM2, ratio of Bcl-2/Bax, PARP, and caspases;2. Histological experiments:We detected DEK protein and mRNA levels in4fresh colon cancer and paired adjacent normal colon tissues, and used immunohistochemical method in109cases of colorectal cancer paraffin-embedded specimens to evaluate the DEK protein expression. The relationship between DEK expression and clinico-pathological characteristics, the survival rates after tumor removal, and multivariate survival analysis were analyzed to verify the clinical value of DEK protein expression in patient prognosis.Results:1. Cytological experiments:According to the results of RT-PCR and Western assays, both of DEK mRNA and protein level was high in colon cancer cell lines HCT116, HT29, SW480, and SW620. Immunofluorescence results show that DEK gene expression mainly in nucleus of SW620cells. Determined by MTT and colony formation assays after DEK gene silenced by siDEK at100nM, SW620cells proliferation significantly decreased (P<0.05). Hoechst33342staining and flow cytometry results show that, the early-apoptosis significantly increased for siDEK transfected SW620cells. We also observed that DEK depletion decreased SW620cells motility and invasiveness by wound healing assays. We detected the expression levels of mutant-p53/MDM2, Bcl-2/Bax, and caspases, which were related with cell apoptosis in SW620cells knocked down DEK gene, and found that DEK can up-regulate the level of mutant-p53and MDM2, ratio of Bcl-2/Bax, PARP, but down-regulate the level of caspase-3, and caspase-9. Additionally, the loss of DEK in SW620cells resulted in a moderate increase in E-cadherin and β-catenin expression.2. Histological experiments:Both protein and mRNA level in cancer tissues were significantly higher than in no-cancerous tissues by RT-PCR and Western blot assays in4fresh colon cancers and paired adjacent normal colon tissues. The immunohistochemical testing in109cases of colorectal cancer paraffin-embedded specimens showed that, the adjacent normal colorectal mucosa (DEK protein positive rate was33.0%, strongly positive rate9.2%) and52cases of adenoma sample (DEK protein positive rate was32.7%, strongly positive rate13.5%) were lower than which in the colorectal carcinoma (DEK protein positive rate was97.2%, strongly positive rate48.6%). It can be found that the DEK protein overexpression was closely related with patients tumor size, differentiation, lymph node metastasis, serosal invasion, histopathological staging. Moreover, disease-free survival and5-years survival rates of the patients with DEK protein overexpression were significantly lower than which of the others, and the patients with DEK overexpression combined lymph node metastasis, serosal invasion, later stage, and CEA level had lower5-years survival rate than the patients with DEK overexpression but without the other factors. DEK protein overexpression was an independent risk factor (HR:1.805, P=0.004) for the prognosis of patients with colorectal carcinoma.Conclusions:Oncogene DEK highly expressed in colorectal carcinoma cells and tissues; DEK exacerbated the proliferation, colony formation, and migration of colorectal carcinoma cells; DEK inhibited cell apoptosis through preventing the activity of mitochondrial pathway; DEK promoted the migration of colorectal carcinoma cells involving in E-cadherin/β-catenin inhibited. DEK overexpression was an independent risk factor for the prognosis of patients with colorectal carcinoma.