| Gliomas(gliomas)are the most common primary malignant tumors in the central nervous system,especially in high-grade gliomas.Although a combination of surgery,chemotherapy,and radiotherapy has been adopted,the postoperative High recurrence rate and drug resistance make it difficult to effectively cure tumors.These characteristics are closely related to the high proliferation and high infiltration growth of tumor cells.Homeobox protein cut 1(CUX1)contains three subtypes with different structures,and studies have shown that the three subtypes are involved in the development of multiple types of malignant tumors.However,the expression of CUX1 subtype in glioma and the specific mechanism of action are still unclear.Here,a series of experiments proved that knockdown of P75CUX1 inhibited the migration and invasion of glioma cells in vitro and in vivo.In the discussion of the mechanism,it was observed that P75CUX1 promotes the proliferation,migration and invasion of glioma cells by activating theβ-catenin-mediated epithelial-mesenchymal transition(EMT)process.In addition,CUX1 has also been proven to promote the progression of glioma through a variety of other signaling pathways(such as Hippo and PI3K/AKT).Chapter 1 The expression of CUX1 in glioma:methods:(1)Through the analysis of public databases,obtain the expression of CUX1mRNA in different types of glioma samples.(2)Using Western blot analysis to distinguish the expression of different CUX1 subtypes in glial cells and different tumor cell lines(3)Use IF and IHC staining to verify the cells and clinical samples to prove the relationship between the expression location,expression level and pathological grade of CUX1 in gliomas.results:(1)Through the analysis of public databases,it is found that CUX1mRNA is overexpressed in gliomas.(2)From the WB results,the expression of P75CUX1 in glioma tissues was significantly higher than that in non-tumor tissues.(3)The results of IF and IHC show that P75CUX1 is only expressed in the nucleus and has a high protein level in glioma tissue.The expression level in glioma tissue is also significantly higher than that in glial cells,and is related to pathological grades.It is positively correlated.Chapter2 P75CUX 1 knockdown inhibited cell migration and invasion but not proliferation in vitromethods:(1)First,use siRNA and shRNA vectors to knock down the expression of P75CUX1 in U251 and U87 cells transiently and stably,and use WB analysis to confirm the efficiency,and then perform EDU,CCK8,Transwell,Boyden,and scratch experiments to verify the effects of CUX1 on cell proliferation,invasion,The influence of migration ability determines the function of P75CUX1 in glioma cells.results:(1)In the scratch experiment,Transwell,and Boy den experiments,it was observed that the tumor cells knocked down by P75CUX1 significantly reduced the ability of migration and invasion,while in the experiments of EDU and CCK8,it was observed that the tumor cells knocked down by P75CUX1 had no proliferation ability.Chapter3 P75CUX1 knockdown inhibited cell migration and invasion in vivomethods:(1)U87 cells transfected with shCUX1 or shCtrl were planted in the brains of 10 nude mice,the mice were euthanized,and the brains were surgically removed 20 days later for hematoxylin-eosin(HE)and immunohistochemistry(IHC)staining(2)In order to explore the molecular mechanism of P75CUX1 promoting glioma infiltration,WB was used to detect the expression levels of EMT-related proteins and MMPs.results:(1)The results of HE and IHC showed that the in situ tumor formed in the control group was more aggressive,while the tumor formed in the experimental group was the opposite.(2)E-ca and Cluadin were detected in U87 and U251 cells knocked down by P75CUX1,and the expression of ZO-1 was significantly increased,while the expression of N-ca,Slug,Snail,β-catenin,MMP2,and MMP9 reduce.At the same time,IHC results of intracranial tumor xenograft models also confirmed that knockdown of P75CUX1 significantly down-regulated the expression of EMT-related proteins,such as N-ca,Slug,β-catenin and MMPs.Chapter4 P75CUX1 regulated β-catenin expression and activity in gliomamethods:(1)Using bioinformatics methods to predict the possible biological functions of CUX 1 in gliomas,and the relationship between CUX 1 mRNA expression and βcatenin mRNA expression levels.(2)The relationship between the expression of CUXl and β-catenin and the activity of β-catenin was verified by WB,IF and tissue IHC.results:(1)The expression of CUX 1 mRNA has a significant correlation with the activity of Wnt/β-catenin pathway.After analyzing the mRNA data of glioma and NB tissues in the database,it was observed that there was a significant positive correlation between the mRNA expression levels of CUX 1 and β-catenin(2)The WB,IF and tissue IHC experimental results of the cell line showed that P 75CUX1 knockdown can significantly inhibit the expression of β-catenin mRNA,thereby further inhibiting the activity of the Wnt/β-catenin signaling pathwayChapter5 P75 CUX 1 regulated glioma cell EMT,migration,and invasion mediated via β-cateninmethods:(1)Use β-catenin plasmid to transfer into CUX1 knockdown U87 and U251 cells to restore β-catenin expression,analyze the expression of EMT-related proteins by WB,and analyze the invasion and migration by Transwell and Boyden experiments.results:(1)In U87 and U25 cells knocked down by CUX1 with β-catenin plasmid,the expression of EMT-related proteins was up-regulated,and it was observed in Transwell and Boyden experiments that the migration and migration of U87 and U251 cells knocked down by CUX1 was observed.Invasive ability is restored by exogenous β-catenin.Chapter6 P75CUX1 affected several signaling pathways in gliomamethods:(1)Using bioinformatics analysis and WB methods to explore how P75CUX1 affects the migration and invasion of gliomas through a variety of different signal pathways.Results:It is proved that P75CUX1 can not only activate Wnt/β-catenin pathway,but also activate other pathways,such as Hippo and PI3K/AKT pathway,to promote the EMT process of glioma and enhance its invasion and migration ability. |
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