Mechanism Of ITGB1 And Its Role In Promoting The Proliferation Of Glioma Cells

Malignant gliomas represent the most common type of brain tumor in adults with an annual incidence of 5 per 100,000 individuals.According to the World Health Organization(WHO)guidelines,they can be characterized as astrocytomas,oligodendrogliomas,ependymomas,or oligo-astrocytomas(mixed gliomas).Another WHO classification is based on the malignancy of the neoplasms and ranges from grade I(low-proliferative noninvasive tumors)to grade IV(highly invasive,mitotically active,and necrosis-prone glioblastoma multiforme(GBM,malignant glioma).Most of the diagnosed GBMs(90–95%)are primary tumors.Medulloblastomas,the most frequent neoplasms in children,are of a cerebellar origin and are therefore not included in this glioma classification system.In the treatment of malignant gliomas,the best treatment is to maximize surgical resection,combined with radiotherapy and adjuvant chemotherapy,but in the past ten years,the effect of the treatment is minimal.The median survival time of patients with glioma after surgery was 1.8 years.Even tough there have been exploring effective treatment methods to improve the therapeutic effect of glioma both at home and abroad,including in radiotherapy and chemotherapy,photodynamic therapy and immunotherapy,but ultimately unrewarding,statistical survival rate has little progress compared with it,which was 17-24% 5 years ago.Researches on genes and molecular biological phenotype of gliomas are important.Currently,gene mutations play a key role in cell proliferation,cell apoptosis,migration and angiogenesis of malignant gliomas.There are many studies have demonstrated that Nocth signaling pathway has key function of modulating the development and progression of gliomas.Nocth receptors can regulate the activity of the main cells in the process of development in embryonic phase and postpartum.Some downstream molecular modulators of Notch pathway can promote MSCs differentiated in specific direction by activating Notch.Notch can prevent neural cell differentiation by maintaining neural stem cells function and promoting neural precursor cells enter into glial cells lineage.It could upregulate the expression of Nocth receptors in glioma,which has been proofed its tumorigenicity in vivo and vitro.Mammalian genome encoding four Notch homologous genes(Notch1-4),which were binded with five ligands(Deltex 1/3/4,Jagged1/2)of Nocth pathways.There are multiple Notch ligands in vertebrate animal cells.Among them,ones with Deltex homologous called the Deltex or the Deltex-like.Notch signaling pathway can be divided into the CSLdependented and non CSL-dependented.Recent studies have found that Deltex protein plays an important role in mediating non CSL-dependented Notch pathway.Studies have shown that Deltex-1 binds with ANK and positively regulate Notch signal.However,overexpression of Deltex can inhibit the Notch pathway.Studies have shown that Deltex-1 could negatively regulate Notch through combining with intracellular ANK repeat sequences.If the Deltex-1was overexpression,the effect of Notch signal would be inhibited.This research has mainly used gene chip technology and bioinformatics analysis to screening of genes expressing significantly differences from gene chip,which has been collected by GCBI.Then detect genetic screening in glioma clinical tumor specimens and the normal control group,the differences between analysis of the relationship between genetic and clinical prognosis of glioma.In addition,use lentiviral vector technology,overexpressing and interfering expression of the differential gene in glioma cells,to observe their effects on glioma cell proliferation.Part? Analysis of genes associated with activated Nocth pathway by using biological information science databaseObjectives: To screen genes that may play key role in activating Nocth pathway.Methods: The analysis was performed on GCBI.We selected an microarray data form GCBI and used it for pathway analysis,gene ontology analysis and gene coexpression network analysis.Results: 1.According to the database from GCBI,microarray(No.: GSE22772)was selected.Gene microarray technology can detect 7962 effective m RNAs with fold changes>1.1 in Deltex-1 knock-down 87 cells.2.According to the analysis of associated pathways,10 pathways which have strong relationship with Deltex were found.Activation of Nocth pathway may involving multiple biological process including cell apoptosis,cell cycle and signal transduction.3.Then we focus on the intersection of gene functions and associated pathway and found a key modulator: ITGB1.Conclusion: According to the analysis of database GSE22772 combined with GO analysis and gene coexpression analysis,ITGB1 may paly an important role in activated CSL-independent Notch signaling pathway.Part? Expression of ITGB1 in gliomas and its relationship with prognosis of patientsObjectives: 1.To detect the expression level of ITGB1 in clinical specimens and glioma cell linces.2.To analyze the association of ITGB1 and prognosis of gliomas.Method: 1.m RNA and protein expression levels of ITGB1 in clinical glioma tumor samples and tumor adjacent normal tissues were detected by real-time fluorescence quantitative PCR and immunohistochemistry.2.Retrospective case and follow up,then the relationship between the expression of ITGB1 and the prognosis of patients with gliomas were analyzed by using Kaplan-Meier curve.Results: 1.The average m RNA expression level of ITGB1 in 24 clinical glioma tumor samples were higher than that of tumor adjacent normal tissues,and the difference was statistically significant(P<0.05).The results of immunohistochemistry showed that the expression of ITGB1 protein in tumor tissue was significantly higher than that in normal tissue,and the difference was statistically significant(P<0.05).2.Through the screening of 43 cases of patients,ones expressing less ITGB1 had longer survival time,high expressions of ITGB1 in patients were on the contrary.Conclusion: ITGB1 in glioma tissues was significantly up-regulated,and negatively correlated with the survival time of patients,can be used as a potential biomarker to predict patient prognosis guiding the clinical diagnosis and treatment.Part ? The effect of ITGB1 on the proliferation of glioma cells and the relationship with the Notch signaling pathwayObjectives: To explore the effect of ITGB1 on proliferation of glioma cells,and its relationship with Notch signaling pathway.Method: 1.The m RNA expression of ITGB1 in glioma cells(U251?SHG44?U373?U87?T98G)was detected by real-time fluorescence quantitative PCR,and then the lowest and highest cell lines were selected for follow-up experiments.2.Sh-ITGB1 plasmid was transfected into U87 cells with lentivirus to ensure the expression of ITGB1 interfered efficiently.Oe-ITGB1 plasmid was transfected into U251 cells with lentivirus to ensure the expression of ITGB1 overexpressed efficiently.3.After interference and over expression of ITGB1,MTT assay was used to detect the change of proliferation in glioma cells.4.After interference and over expression of ITGB1,colony formation assay was used to detect the change of clonality in glioma cells.5.After interference and over expression of ITGB1,Western blot was used to detect protein expression of Notch and Hey1,which is the pathway downstream protein of Notch signal pathway,for exploring the relationship between ITGB1 and Notch pathway.Results: 1.q RT-PCR showed that there was a different expression among five kinds of glioma cell lines in m RNA expression of ITGB1,U87 expression was the most,U251 expression was the lowest.2.Fluorescence microscopy confirmed that lentivirus transfected successfully into glioma cells.q RT-PCR and western blot respectively verified that overexpression and interference expression of ITGB1 in U251 cells and U87 cells was efficient(P<0.05).3.MTT assay suggested the proliferation of U87 was significantly inhibited after interference ITGB1,and the proliferation of U251 was significantly promoted after ITGB1 overexpressed,the difference had statistical significance(P<0.05).4.Clone formation array showed that the interference of ITGB1 significantly inhibited U87 clone forming ability,overexpression of ITGB1 significantly promoted U251 clone forming ability(P<0.05),the difference has statistical significance(P<0.05).5.After ITGB1 decreased,expression of Nocth protein and Hey1 protein also appeared significantly decreased(P<0.05);overexpression of ITGB1 significantly up-regulated Notch protein and Hey1 protein(P<0.05).Conclusion: ITGB1 can significantly promote the proliferation of glioma cells.And the ITGB1 and Notch signaling pathway can form feedback regulation mechanism.