Role And Mechanism Of PIM1 In The Tumorigenesis And Progression Of Glioma

Glioma is the most common intracranial primary tumor with high morbidity and recurrence rate and poor prognosis.Currently,the main clinical treatment for surgical resection combined with postoperative radiotherapy and chemotherapy,however,patients still difficult to obtain a longer survival.Therefore,to study the molecular mechanism of glioma,to find a new target for the treatment of glioma has become one of the hot spots in the diagnosis and treatment of glioma in recent years.The human Pim-1 gene encoding a serine/threonine kinase(serine/threonin ekinase):Pim-1,which is widely expressed in various tissues and cells,and p lays an important role in cancer development.Pim encoding filamentous/thre onine kinase of proto oncogene,by Pim-1,Pim-2 and Pim-3 family members of the Pim family is a calcium/calmodulin dependent protein kinase family,its expression in different tissues of different height:the expression of Pim-1 in hematopoietic cells;Pim-1 kinase potential tumorigenicity in blood and sol id tumors is highly expressed.Targeted inhibition of protein kinase can signifi cantly improve the survival time of patients with glioma.The results show th at VEGF/AKT/eNOS signal pathway glioma cell survival,proliferation,invasio n,apoptosis and angiogenesis in the process,so far,the expression of PIM1 i n glioma and normal brain tissues difference is not clear Further study is nee ded for PIM1 mediated proliferation,invasion and angiogenesis of glioma cell s and the abnormal activation of VEGF/AKT/eNOS signaling pathways.In this study,we first examined the expression level of PIM1 in normal brain tissue and different pathological grades of glioma tissues;next,we studied the gene knockout of glioma cell proliferation after PIM1,the ability of invasion and angiogenesis;finally,we investigate the proliferation of VEGF/AKT/eNOS signaling pathway in the regulation of PIM1 glioma cells the invasion and angiogenesis mechanism in the process.Part One PIM1 expression in glioma and non-neoplastic brain tissues Objective:To investigate the expression of PIM1 in glioma of different malignancy and non-neoplastic brain tissues.Methods:Western Blot and RT-qPCR were used to detect the expression of PIM1 protein and mRNA in 28 cases of glioma tissues and 15 cases of damaged normal tissues removed by decompression in the neurosurgery department of Wuhan General Hospital.The expression of PIM1 protein and mRNA were detected by t test.Single factor ANOVA and chi-square test were statistically significant(P<0.05).Results:PIM1 protein expression in the glioma normal brain tissue(P<0.05).The mRNA expression of PIM1 in glioma tissues was higher than that in normal brain tissues(P = 0,010)There was no difference in mRNA expression level(P =0.734),indicating that PIM1 mRNA expression had no correlation with the pathological grade of glioma.Conclusion:The expression of PIM1 protein and mRNA in glioma tissue is higher than normal brain tissue,suggesting that PIM1 may play an important role in the development of glioma.Part Two Role of PIM1 in the proliferation,invasion and angiogenesis of malignant gliomaObjective:To investigate whether gene knockdown of PIM1 can affect the proliferation,invasion and angiogenesis of U87MG cells.Methods:The glioma cell line U87MG was used as the target.PIM1 was knocked down and PIM1 was overexpressed by plasmid transfection.The expression of PIM1 protein in U87MG glioma cells was detected by Western Blot.The expressions of CCK8 Proliferation assay was used to detect the proliferation of U87MG cells in each experimental group.Transwell invasion assay was used to detect the invasion ability of U87MG cells in each experimental group.The ability of vascularization of U87MG cells in each experimental group was tested.Using t test,one-way ANOVA statistical analysis,P<0.05 for the difference was statistically significant.Results:After shRNA knockdown of PIM1,the expression of PIM1 protein in U87MG glioma cells was significantly decreased in each experimental group,which indicated that the transfection was successful.The proliferation,invasion and angiogenesis of U87MG glioma cells in each experimental group were decreased P<0.05).The proliferation,invasion and angiogenesis of U87MG glioma cells in each experimental group were enhanced after transfection with plasmid p1(P<0.05).Conclusion:Our result showed knockdown of PIM1 by shRNA lead to the glioma cell proliferation and invasion capability reduction and overexpression of PIM1 significantly increased the glioma cells proliferation and invasion capability in vitro.These results provide evidence that PIM1 play a crucial role in glioma cells proliferation and invasion.Part Three Effect of PIM1 on VEGF/AKT/eNOS signaling pathway of malignant glioma cell line U87MGObjective:To explore the role and Mechanism of VEGF/AKT/eNOS signaling Pathway in Glioma cells.Methods:The glioma cell line U87MG was used as the object of study.The experiment group:the gene knockout PIM1 experiment with shRNA interference was divided into three groups:normal blank control group.Western Blot was used to detect the expression of PIM1、AKT、p-AKT、VEGFA、eNOS、p-eNOS、EGFR protein in malignant U87MG glioma cells of each experimental group.The statistical analysis was performed by t test and univariate analysis of variance(P<0.05).Results:After shRNA interference technique knocked out PIM1,the expression of PIM1 protein in U87MG malignant glioma cells in interference group decreased significantly,indicating that the transfection was successful.There was no change in the total protein level of AKT,p-eNOS in U87MG malignant glioma cells in the interference group,while the expression of VEGFA,p-AKT,eNOS protein decreased in the malignant glioma cells of U87MG(P<0.05).Conclusion:PIM1 may be involved in the activation of VEGF/AKT/eNOS signal transduction pathway in glioma cells.This study is expected to find a new target and breakthrough for the treatment of malignant gliomas.