| Globally,the incidence of cancer in the central nervous system is 2.6-37/100,000inhabitants per year.Among brain malignant tumors,diffuse glioma is the most common,accounting for about 30%of primary brain tumors originated from glial cells.Astrocytomas are the most common gliomas in humans,accounting for 80 percent of central nervous system malignancies-According to the WHO classification of the central nervous system tumors in 2007,diffuse astrocytoma infiltrating type can be divided into three:diffuse astrocytoma(WHO Ⅱ),anaplastic astrocytoma(WHO Ⅱ),glioblastoma(WHO Ⅳ).The average overall survival rate of glioma patients was about 20 to 36 months,while the survival rate of patients with the highest degree of malignancy was never more than 14 months.In cases of recurrent glioma,the average time for tumor progression was only 8 weeks,despite aggressive treatment.The current treatment of gliomas is surgery combined with chemotherapy and/or radiation.Over the past decade,neurosurgeons have used advanced intraoperative imaging methods,fluorescence based tumor biomarkers and real-time mutation analysis to maximize the extent of tumor resection.In addition,the use of stimulation-mapping techniques,corticospinal tract imaging,and stereotactic thermal ablation continues to improve and minimize perioperative morbidity.However,many patients with glioma still show high resistance to these interventions,and relapse is frequent.Therefore,it is of great significance to search for the molecular mechanism of glioma occurrence and development and to seek new therapeutic targets.KIF20A,also known as RAB6KIFL/MKlp2,is a member of the kinesin6 family,a microtubule-related motor protein that was initially found to be located in the golgi body and plays an important role in the organelle by interacting with Rab6 in the form of GTP binding.Cytokinesis is performed in the last step of mitosis.It also transports retrograde vesicles from the golgi apparatus to the endoplasmic reticulum during the intercellular phase.KIF20A protein is controlled by the E2F-retinoblastoma protein p16 pathway,and its widely expressed mRNA exists in fetal and adult proliferative tissues.KIF20A belongs to a large class of motor proteins that accumulate in mitotic cells.Microinjection of anti-kif20a antibodies will cause cell division failure.It is reported that the abnormal function of KIF20A,a member of the driving protein family,plays an important role in the occurrence and development of tumors.Overexpression in multiple tumors has been shown in studies to significantly inhibit the growth of tumor cells by inhibiting the expression of KIF20A in pancreatic cancer cells by small interfering RNA.In addition,KIF20A promotes the vitality and invasiveness of pancreatic cancer cells by transporting human insulin-like growth factor 2-mrna binding protein 3(IGF2BP3)and IGF2BP3 binding transcript along microtubules to the cell processes.The expression characteristics of KIF20A in various tumors indicate that it may be a potential target for tumor diagnosis and treatment.Studies have shown that KIF20A is a new tumor-associated antigen(TAA),a potential target for anticancer immunotherapy in pancreatic cancer cells that express KIF20A,at least when human leukocyte antigen-a2(HLA-A2)is limited.However,the expression of KIF20A in glioma,its relationship with cell function,tumor biological behavior and corresponding potential molecular mechanisms have not been reported.This study aims to explore the relationship between KIF20A expression in glioma and its clinical and pathological features.The effects of KIF20A on proliferation,migration,invasion,apoptosis and cell cycle in glioma cells were studied.And then the related molecular mechanism is preliminarily discussed.It provides new theoretical basis and new direction for gene targeting therapy of glioma.PART ONE Expression of KIF20A in human glioma and its correlation with clinical pathological featuresObjectiveThe expression of KIF20A in human glioma tissues and normal brain tissues was detected,and the correlation analysis was conducted by combining the clinical and pathological parameters of the patients,to explore the expression of KIF20A in glioma tissues.Methods94 human astrocytoma specimens were collected from QiluHospital of Shandong University,and 10 normal brain tissue specimens were from patients undergoing decompressive osteotomy in the second people’s hospital of Liaocheng.All specimens were confirmed by two pathological experts.Immunohistochemistry was used to detect the protein expression of KIF20A in glioma and normal brain tissue,and the correlation analysis was conducted by combining clinical pathological parameters.Patients were followed up and survival data were collected for COX univariate and multivariate survival analysis.Western blot was used to detect the expression of KIF20A protein in four glioblastoma cell lines,and to screen suitable cell lines for subsequent cell function tests.Results1.Immunohistochemical results indicated that KIF20A was expressed in malignant astrocytomas.Smooth muscle tissue was a negative control.It was found that 65 out of 94 cases of malignant astrocytomas were positive for KIF20A staining,and only 1 out of 10 cases of normal brain tissue was positive for staining.2.The expression of KIF20A in glioma is independent of the patient’s age,gender and tumor size,but significantly correlated with the WHO pathological grade of glioma.With the increase of pathological grade,the staining intensity and positive rate of KIF20A increase.3.Univariate survival analysis and multivariate analysis showed that high expression of KIF20A was correlated with poor overall survival(P= 0.022;Risk(HR)=1.925;95%confidence interval(CI)=1.098-3.375.Kaplan-meier survival curve was plotted,and log-rank analysis showed that KIF20A overexpression was significantly correlated with the prognosis of glioma patients.4.In addition,Western blot detection of KIF20A expression in glioblastoma cells showed that KIF20A expression was much higher in U87 and U251 cells than that in T98 and LN229 cells.U251 and U87 cells were screened for biological behavior in vitro.ConclusionsKIF20A was overexpressed in glioma.The expression intensity and range of KIF20A in human glioma were enhanced with the increase of WHO’s pathological grade,indicating that KIF20A was closely related to the malignant degree of glioma.High expression of KIF20A was shown to be an independent prognostic factor for overall survival in glioma patients.PART TWO The effect of KIF20A on the biological behavior of human glioma cell linesObjectiveThe biological behavior of KIF20A on proliferation,migration,invasion and apoptosis of human glioma cells and its effect on phase distribution during cell cycle were analyzed by in vitro experiments.MethodsGlioblastoma Cells U251 and U87 were screened for biological behavior experiments in vitro.By transfecting siRNA,KIF20A expression was disturbed,and the interference effect was detected by quantitative real-time PCR and Western blot.On this basis,CCK8 was used to detect the changes of growth activity of the two cell lines and observe the differences of clone formation.Transwell test was used to detect the changes of migration and invasion of glioma cells.The effects of cell cycle phase distribution and apoptosis of glioma cells were measured by flow cytometry.Results1.Rt-qpcr and western blot analysis showed that in U251 and U87 cells,compared with the control group,siRNA KIF20A significantly decreased the expression of KIF20A in the interference group at mRNA and protein levels.2.CCK8 was used to analyze the cell growth activity,showing that the OD value of the interference group was lower than that of the control group.In addition,knockdown of KIF20A expression in U251 and U87 cells reduces the number of clones.3.Transwell experiment results showed that interfering KIF20A expression significantly reduced the migration and invasion ability of the two cells.4.Flow cytometry showed that the percentage of G0/G1 cells in U251 cells increased from 48.74 ± 1.33%to60.26 ± 1.27%.The percentage of G0/G1 cells in U87 cells increased from 48.33 ± 0.70%to61.49 ± 3.55%.However,the percentage of s-phase cells in U251 cells decreased from 35.53 ± 1.85%to24.40± 0.41%.The percentage of s-phase cells in U87 cells decreased from 38.08 ±0.33%to 24.76 ± 2.16%.5.After interference of KIF20A,the total apoptosis rates of U87 and U251 cells were increased compared with the control group.ConclusionsIn vitro knockdown of KIF20A inhibited the activity and proliferation of glioblastoma cells,down-regulated KIF20A induced cell cycle arrest at G0/G1 stage,induced apoptosis of glioblastoma cells and inhibited their migration and invasion.PART THREE Molecular biological mechanism of KIF20A to promote the proliferation and apoptosis of human glioma cellsObjectiveBy studying the expression of KIF20A in glioma tissues and cells and the influence of KIF20A on the biological behavior of glioma cell lines in vitro,relevant molecular biological mechanisms and cell signaling.pathways were searched for new perspectives of glioma treatment strategies.MethodsAfter western blot detection of interfering KIF20A expression,changes in protein levels of PI3K-AKT signaling pathway,NF-κB signaling pathway and p53signaling pathway,as well as changes in expression of cell cycle regulation protein and apoptosis related protein were performed and statistically analyzed.Results1.Interference with the expression of KIF20A in glioma cells,compared with the control group,the results showed that the expression levels of NF-expressed B and P53 proteins in the signaling pathway were not changed,but the expression levels of phosphorylated AKT(pAKT)were decreased.2.Knocking down the expression of KIF20A can reduce the expression of c-myc and CyclinD1 cyclin-related proteins.3.In addition,KIF20A knockdown can significantly reduce the expression of apoptotic protein BCL2 in U251 and U87 cells,while increasing the expression of BAX.ConclusionsIt was indicated that after interfering with the expression of KIF20A,the survival ability of glioma cells was reduced,and the proliferation of glioma cells was inhibited.These effects might be achieved by inhibiting the PI3K/AKT signaling pathway.The decreased expression of KIF20A reduced the expression of cell cycle regulation proteins c-myc and CyclinD1,and prompted glioblastoma cells to be blocked at G0/G1 phase instead of G2/M phase.KIF20A was down-regulated to induce apoptosis by inhibiting Akt activity and activating endogenous apoptosis pathway,namely regulating the proportion of BCL2/BAX. |
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