Effect Of Low Expression Of CENP-W On Human Glioma U87 Cells

Background:Mechanism of tumor development has been a difficult problem to medical community. Many studies of pathogenesis is still in its infancy and the exploration of cerebral glioma was never stopped. Traditional treatments to glioma methods include surgery, chemotherapy and radiotherapy, or a combination of surgery and chemotherapy. But the effect is not good and the cure rate and low recurrence rate are no significant improvement. In the occurrence and development of glioma, the abnormal expression and treatment of related genes have become a research hotspot.Cancer is considered to be a disease caused by the cell cycle.The main characteristics are the performance of cell-cycle disorders and uncontrolled proliferation.Centromere plays an important role in the process of mitosis. Centromere is mainly composed of DNA binding protein composition and the protein was called centromere proteins which are involved in the process of cell division together with the assembly of the centromere. Centromere can ensure proper separation of mitotic chromosomes.Centromere protein W,also as CUG2,was was an oncogene was found in 2007 by Soojin Lee. CENP-W located on chromosome 6q22.32, genomic DNA is about8.5kb, contains three exons, cDNA is about 531 bp, mRNA is about 600 bp, a protein of about 10 kD. Further research Nishino, T found that centromere group to form a stable tetraploid CENP-T-W-S-X structure, and with the DNA and form a positive spiral structure wound 100 bp of DNA forming a similar nucleosomes.Overexpression of CENP-W can promote cell proliferation, on the other hand can induce cell apoptosis, whice has a very complicated mechanism. It has been reported in the literature that CENP-W in most of the human tumors in different parts showed high expression and few showed low expression. However, the current study about CENP-W reported very less. CENP-W impact on tumor cell function has not been reported. In this study, U87 glioma cells were treated as experimental subjects,which was transfected with CENP-W-siRNA to downlowed CENP-W in human glioma U87 Cells. Exploring the relationship between CENP-W and U87 cells in vitro by a variety of experimental methods, hoping to provide some experimental basis for the treatment to glioma.Objective:To explore the effect(such as Proliferation,Invasion,Cell cycles,and so on) of low expression of CENP-W on human glioma U87 cells.Methods:1. Preparatory work, cell recovery, cell culture, cryopreservation.2. The siRNA was transfected into U87 cells by lipofectamine2000 to inhibit the expression of CENP-W. After transfection 6h, the cells were placed under an inverted fluorescence microscope and transfection efficiency was observed. Punctate fluorescence can be seen in cells, if the proportion of fluorescent cells accounted for more than 50 percent is judged to be successful transfection.3. The expression of CENP-W mRNA and protein were examined by real time fluorescence quantitative PCR(FQ-PCR) and Western blot.4. MTT assay method and Brdu experment were used to detect proliferation inhibition of U87 cells transfected with CENP-W-siRNA. Tablet cloning experiments was used to detect cell proliferation.5. Cell scratch test, Transwell experiments were used to detect the migration and invasion of U87 cells transfected with CENP-W-siRNA.6. The apoptosis and cell cycle of cells were detected by flow cytometry(FCM).Results:MTT assay, plate clone experiment, Brdu experiment showed that the proliferation, colony of the experimental group transfection with CENP-W-siRNA is lower significantly than the control group and negative control group. Transwell experiment and scratche experiment showed that the migration and invasion of U87 cells transfection with CENP-W-siRNA decreased significantly than the control group and negative control group. Flow cytometry analysis showed that the cell cycle of U87 cell transfected with CENP-W-siRNA was arrested in G0 / G1 phase and cell proliferation is limited. At the same time the apoptosis of test group cells is increased than the control group and negative control group(P<0.05).Conclusion:Downregulation CENP-W can inhibit the proliferation、migration、invasion and promote apoptosis of glioma cells,which can explain that CENP-W may play an important role in the development of glioma in a certain extent. CENP-W may serve as a potential target of gene therapy for glioma.