| Glioma arises from glial cells and is the most frequent intracranial tumor.The incidence of glioma in the last 3 decades is increasing by 1-2%annually.Due to the development of diverse treatments for glioma,including image technology,surgery,radiotherapy,chemotherapy and targeted therapy,the 2-year survival rate of glioma patients arises from 10.4%to 26.5%.However,the outcome of glioma remains poor.Currently,the1-year and 5-year survival rate for adult high-grade glioma is 30%and 13%,respectively.The complex genic mutations of glioma cells lead to the resistance and recurrence of glioma.Thus,investigations of the tumorigenesis and development mechanism of glioma not only promote the understanding of its biological behavior,but also provide theoretical basis for new treatment strategy.Epidermal growth factor receptor(EGFR)plays important roles in the development of giloma.The high expression of EGFR not only promotes proliferation,invasion and migration of glioma cells,but also inhibits their apoptosis.Meanwhile,it also promotes the angiogenesis of tumoral tissues.EGFR variant?(EGFRv?)is the most frequent variant of EGFR.It is expressed in more than 40%of giloma,but rarely expressed in normal human tissues.The activity of EGFRv?associated enzymes plays key roles in the dysfunction of signal transduction pathway and cell cycle arrest.Centrosomal protein 55(CEP55)is a newly characterized mitotic phosphoprotein that plays important roles in cell division.The overexpression of CEP55 causes defects in cell division which lead to the tansformation of normal cells into unstable multinuclear cells.Recent studies confirm the high expression of CEP55 in several human tumors including colon cancer,breast cancer and lung cancer.The abnormal expression of CEP55 is closely related to the tumoregenesis and poor prognosis of these tumors.However,little is known about the expression of CEP55 and its functions in human glioma.Objectives1.To clarify the expression of CEP55 in gliomas and normal human brain tissues.2.To investigate the effect of CEP55 on the proliferation of human glioma cell line U251.3.To elucidate the effects of EGFRv ? on CEP55.4.To verify the effect of EGFRv? and CEP55 on the progression of glioma in vivo.Methods1.The expression of CEP55 in gliomas and normal human brain tissues1.1 The expression of CEP55 protein in gliomas and normal human brain tissues by Western-blotTissues were ground and dissolved in the mixture of RIPA and PMSF.The supernatants were collected after centrifugation.Then the protein was transferred to the PVDF membrane and incubated with anti-CEP55 antibody at 4?overnight.This was followed by incubation with HRP-conjugated secondary antibodies.Bound proteins were visualized by chemiluminescence.1.2 The expression of CEP55 gene in gliomas and normal human brain tissues by q PCRThe total RNA of 40 cases of glioma tissues and 10 cases of normal human brain tissues were extracted and reversed transcribed to c DNA.qPCR was performed to detect the expression of CEP55 gene.1.3 The association of CEP55 expression and patients'survival was analyzed by Firebrows.2.The effect of CEP55 on the proliferation of human glioma cell line U2512.1 The expression of CEP55 gene in U251 cells by qPCR and Western-blotThe expression of CEP55 gene and protein in U251 cells was determined by q PCR and Western-blot,respectively.2.2 The transfection of CEP55 RNAi vector into U251 cellsThe CEP55 RNAi vector was purchased from Shanghai Genechem Company.The sequence of CEP55-siRNA is 5'-GTGGGAAAGGAAAGCTGAC-3'.The CEP55 RNAi vector was transfected into U251 cells.2.3 The silence effect of CEP55 RNAi on U251 cells detected by q PCR and Western-blotU251 cells were transfected with CEP55 RNAi and screened by toxic puromycin.Then the cells were collected and the CEP55 gene was determined by RT-PCR and the CEP55 protein was determined by Western-blot.2.4 The proliferation of transfected U251 cells detected by CCK-8 kit and edu detectionCells from transfection group,non-transfection group and control group were plated at3000/well in the 96-well plate,respectively.The culture medium was replaced every 3 days.The proliferations of cells in different groups were detected by CCK-8 kit according to the manufacturer's instructions.As for edu detection,cells from different groups were plated in the 6–well plate at 2×10~5/well and incubated at 37?overnight.2?l edu solution was added to the culture medium and the cells were incubated for another 6 hours.The proliferation of cells was detected by flow cytometry according to the manufacturer's instructions.3.The effects of EGFRv? on CEP553.1 The construction of U251 cells that express EGFRv ?The EGFRv?-expressing vector was purchased from Shanghai Genechem Company.U251 cells were separated into 3 groups,namely transfection group,non-transfection group and control group.Three days after transfection,the cells was observed and screened under the fluorescence microscope.Then the cells were collected.3.2 The transfection effect of EGFRv? detected by q PCR and Western-blotThe transfection effect of EGFRv? in U 251 cells was verified by RT-PCR and Western-blot.3.3 The proliferation of U251 cells with high-expression of EGFRv? was determined by CCK-8 kit3.4 CEP55 expression of U251 cells with high-expression of EGFRv ? was detected by qPCR and Western-blot.3.5 U251 cells with high-expression of EGFRv ? and low expression of CEP55 were constructed as previous described.The proliferation of these cells was determined by CCK-8 kit and edu detection.4.The effect of EGFRv ? and CEP55 on the progression of glioma in vivo4.1 The groups of animalsTwenty-five nude mice were divided into five groups,i.e.LV-EGFRv?group,CEP55RNAi-LV group,si-CEP55 group,negative control(NC)group and control group.4.2 The culture of U251 cellsU251 cells for different groups were generated as previous described and cultured in vitro and then collected to make cell suspensions.4.3 The establishment of subcutaneous tumor model of nude miceThe cell suspensions from different groups were injected subcutaneously into nude mice from corresponding groups.4.4 The measurement of tumor sizeThe length and width of tumors were measured every two days since day 7 after inoculation until day 24.Then the mice were sacrificed and the tumors were seperated.The sizes of seperated tumors were measured.Results1.The expression of CEP55 in gliomas and normal human brain tissues1.1 Western-blot results revealed a higher expression of CEP55 protein in glioma tissues than in normal human brain tissues.However,there was no difference in the expression of CEP55 in glioma tissues from different grades.1.2 qPCR results revealed an increased expression of CEP55 gene in glioma tissues from both high-grade and low-grade glioma.And no difference in the expression of CEP55was found between high-grade and low-grade glioma.1.3 Low patients'survival time is associated with high expression of CEP55.2.The effect of CEP55 on the proliferation of human glioma cell line U2512.1 CEP55 expression in U251 cells was verified by Western-blot and qPCR.2.2 Western-blot results revealed that CEP55 protein in transfection group was significantly decreased compared to non-transfection group and control group.RT-PCR results revealed that CEP55 gene expression in transfection group was decreased by more than 95%.2.3 According to CCK-8 detection,compared to non-transfection group and control group,proliferation rate of cells from transfection group was significantly slower;four days later,the proliferation curve of transfection group began to decline while that of non-transfection group and control group sustained.Edu staining detected by flow cytometry demonstrated that the proportion of proliferating cells from transfection group was promptly lower than that of non-transfection group and control group.3.The effects of EGFRv? on CEP553.1 U251 cells expressing EGFRv ? was established by lentivirus vector.The transfection efficiency was verified by Western-blot and qPCR.3.2 Proliferating capacity of U251 cells from transfection group increased since day2,and peaked at day 5.3.3 U251 cells expressing EGFRv? expressed higher levels of CEP55 compared to non-transfection group and control group.3.4 CEP55 expression in U251 cells expressing EGFRv ? was knocked down by RNAi.Then the proliferation of these cells was determined by CCK-8 kit and edu staining.The results showed that the proliferation of U251 cells that expressed EGFRv? decreased when CEP55 was knocked down.4.The effect of EGFRv ? and CEP55 on the progression of glioma in vivoThe tumor sizes of LV-EGFRv?group?NC group and control group were significantly larger than CEP55RNAi-LV group and si-CEP55 group.Conclusions1.CEP55 expression in giloma tissues was higher than in normal brain tissues.2.Low patients'survival time is associated with high expression of CEP55.3.Human glioma cell line U251 expressed high level of CEP55.CEP55 could strengthen the proliferating capacity of U251 cells.4.EGFRv ? may be the upstream regulator of CEP55.5.EGFRv ? enhanced the proliferation of glioma cells by up-regulating CEP55expression,thus promotes glioma progression in vivo.6.CEP55 is a new target for glioma therapy.SignificanceIn this article we report the high expression of CEP55 in glioma.Our in vitro and in vivo data together reveal that CEP55 could enhance the proliferation of giloma cells thus promte the progression of giloma.Besides,we also report for the first time that EGFRv? is the upstream regulator of CEP55.These results further complete the mechanism of glioma progression and provide theroretical evidence and treatment targets for glioma therapy. |
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