Correlation Study Between KNL1 Expression In Glioma And Tumor Microenvironment

Objective:Glioma is the most common primary intracranial tumor,Surgical resection plus radiotherapy and chemotherapy,but the treatment effect was not satisfactory.Immunotherapy is a research hotspot in the oncology.Tumor associated macrophages and microglia,as the intracranial immune cells,are considered to be the key factors in the progress of immunotherapy for glioma.Glioma can induce the "immunosuppressive" phenotype of microglia by secreting chemokines,thus supporting tumor progression.Kinetochore scaffold 1(KNL1)is highly expressed in a variety of tumors and participates in tumor proliferation and apoptosis.However,little is known about its role in glioma and tumor immune response.The purpose of this study is to understand the expression of KNL1 in glioma,explore the relationship between its expression level and survival prognosis,and study the role of KNL1 in tumor microenvironment.Methods:1.The expression of KNL1 and the survival curve of glioma patients were searched in GEPIA.The expression of KNL1 in gliomas with different grades of glioma was inquired in CGGA and TCGA,as well as the related survival curve of patients,so as to analyze the influence of KNL 1 expression on the prognosis of patients;2.The mRNA levels and protein levels of KNL 1 in normal brain tissue and glioma samples were detected by RT-qPCR and IHC;3.Lentivirus LV-KNL1-RNAi was used to stably transfect U87 and U373 cell lines to knockdown KNL1.Meanwhile,the negative control virus LV-NC was transfected.KNL1 expression in shKNL1(experimental group)and shNC(control group)was detected to verify the transfection efficiency by RT-qPCR and Western blotting;4 The expression of a series of chemokines in shKNL1 and shNC was detected by RT-qPCR;5 The chemotactic effect of KNL 1 knockout in glioma U87 and U373 on microglia HMC3 was detected by Transwell;6 The chemokine specific inhibitors UNBS5162 and Pirfenidone were used to reduce the chemokine release of U87 and U373,and the chemotactic effect to microglia HMC3 was detected by Transwell;7 Transwell was used to construct the co-culture model of glioma cells(shKNLl,shNC)with microglia HMC3,and RT-qPCR was used to detect the phenotypic gene differences of HMC3;8 CCK-8 assay detected the proliferation ability of microglia cells co-culturing with glioma after knockdown of KNL1;9 Glioma cells(shKNL1,shNC)were injected into the brain of nude mice by in situ intracranial tumorigenesis technology.The effect of KNL1 in vivo was detected by Hematoxylin-Eosin staining and counting the survival time of nude mice.The difference of microglia phenotype protein was detected by IHC.Results:1 GEPIA data showed that KNL 1 expression in glioblastoma was significantly higher than that in normal brain tissue.According to the database of CGGA and TCGA,pathological grade determines the expression level of KNL 1 in glioma,there is a positive correlation between them,and the higher the expression of KNL 1,the worse the prognosis of glioma patients;2 RT-qPCR and IHC showed that KNL1 expression in glioma was higher than that in normal brain tissue,there is a positive correlation between the pathological grade and KNL 1 levels;3 RT-qPCR and Western blotting showed that KNL1 expression was significantly inhibited in both U87 and U373 cells after lentivirus transfection;4 RT-qPCR showed that the mRNA expression of chemokines decreased in different degrees after knockdown of KNL 1;5 Compared with microglia cultured alone,microglia migration increased in co-culture model.Knockdown KNL1 expression in glioma attenuated the chemotaxis of gliomas to microglia;6 The chemotaxis of gliomas to microglia was significantly reduced by using CXCLs specific inhibitor UNBS5162 and CCLs specific inhibitor Pirfenidone;7 Co-culturing with KNL1 knockdown glioma cells,decreased the expression of M2 polarization phenotype related markers,increased the expression of M1 polarization phenotype related markers,and weakened the proliferation ability of microglia;8 In vivo experiments showed that knockdown of KNL1 significantly prolonged the survival time and reduced the tumor volume in nude mice;9 IHC results showed that the M1 polarization phenotype related markers of microglia were higher than the M2 polarization phenotype related markers in the tumor region after knockdown of KNL1,but the opposite was true without knockdown of KNL1.Conclusion:1 The high expression level of KNL1 is not conducive to the prognosis of glioma patients;2 KNL1 was closely related to tumor growth and survival time;3 Down-regulation of KNL1 could decrease the chemotaxis of glioma to microglia,and drive microglia toward M1 phenotype.