Effect And Mechanism Of NKAP On Glioma

BackgroundGlioma,the most common intracranial tumor in the central nervous system,originates from the neuroectodermal layer and is characterized by a high incidence,high mortality,and high invasiveness.Although there are many studies on gliomas in the world,the analysis of specific pathogenesis has not yet been thorough.Studies have shown that the proliferation,migration and metastasis of glioma cells are closelyrelated to tumor cell receptors,related genes and regulatory molecules.Molecular targeted therapy has obvious advantages in clinical treatment.It has targeted specificity,no cytotoxicity,and has stable effects on cells and has regulatory effects.Current research suggests that,in molecular biology mechanisms,tumor formation is associated with abnormal expression of proto-or anti-oncogenes,and abnormal regulation of cell cycle and apoptosis.Therefore,exploring the genes involved in the regulation of the biological behavior of glioma cells can help further elucidate the pathogenesis of gliomas and provide a clearer and more rational theoretical basis for the gene-targeted therapy of gliomas.NKAP(NF-?B activating protein)is a NF-?B-induced activation factor that is induced by TNF-a and IL-1.It is located in the nucleus and is highly conserved during evolution.Studies have found that NKAP is highly expressed in mature neurons of mouse OB,thalamus,and hippocampus,indicating that NKAP plays an important role in neural development.During the differentiation of T cells,NKAP binds to HDAC3 and CIR,respectively,and becomes part of the Notch transcriptional repression complex,acting as a transcriptional repressor of the Notch pathway.In natural killer T cells(iNKT),NKAP interacts with HDAC3 rather than as a transcriptional repressor of the Notch pathway to regulate the development of iNKT.In addition,NKAP also regulates the proliferation and differentiation of iNKT.We have found that the expression of NKAP is increased in gliomas,but its role and mechanism in tumors have not been studied.Notch signaling interacts with the receptor through the Notch ligand of adjacent cells,and the Notch protein is trimmed three times to release the intracellular Notch receptor domain(NICD)into the cytoplasm,which in turn translocates the NICD into the fine nucleus.The nuclear transcription factor RBP-Jk binds to form the NICD\RBP-Jk transcription complex and regulates the transcription of downstream target genes,thereby producing biological effects.Studies have shown that the Notch signaling pathway is highly conserved in evolution and is inextricably linked to cell proliferation,differentiation,and fate determination.When abnormally expressed,it can easily lead to brain tumors.In the process of tumorigenic transformation of cells,activation of Notch signaling promotes cell proliferation,and at the molecular level,maintains cell size,promotes cell energy uptake and metabolism,activates PI3K/AKt and NF-?B pathways,and inhibits tumor suppression.The oncogene p53 plays an anti-apoptotic role.We cultured glioma cells in vitro and used the experimental techniques of CCK-8,EdU,FACS,Transwell,Matrigel,qRT-PCR,Western Blot,immunohistochemical staining,immunofluorescence staining,and cell transfection to detect NKAP on the brain in vitro.The effects of glioma cell proliferation,migration and invasion,and exploration of the mechanisms by which NKAP exerts its effects.In addition,we also used subcutaneous tumors in nude mice and implanted tumors in nude mice to verify the effects of NKAP on gliomas in vivo.NKAP is expected to become a new site for the diagnosis and treatment of gliomas.Methods1.Exploring the expression of NKAP in glioma tissues and cellsNormal brain tissues and pathological tissues of different grades of gliomas were collected.The expression of NKAP was detected by qRT-PCR,Western blot,and immunohistochemistry.The expression level and pathological grades of gliomas and patients were analyzed.The relationship between prognosis.2.The effect of NKAP on the biological effects of glioma cellsThe lentivirus NKAP-siRNA vector or negative control vector was transfected with U87MG and U251 to establish a stable transfection cell line.The cells were divided into NKAP knockout group(NKAP ko)and control group(NKAP Con).(1)The effect of NKAP on the viability and proliferation of glioma cellsThe transfected cell lines were respectively tested for their ability and proliferation using CCK-8,EdU and FACS assay.(2)The effect of NKAP on the invasion,migration and EMT ability of glioma cellsIn the transfected cell lines,Transwell migration assay,Matrigel invasion assay,and EMT marker expression were used to detect the effect of NKAP on cell invasion,migration and EMT ability.3,The mechanism of NKAP in regulation of gliom via the Notch signaling pathway(1)qRT-PCR technique was used to detect the expression of Notch signaling pathway downstream genes Notch 1,Notch2,Notch3,Notch4,CCND1,HES1,CTNNB1 and DVL2 in the NKAP ko and NKAP Con groups of U87MG and U251 cells,respectively.Further Western Blot experiments were used to further detect the expression of Notch1,and HES1 in the cells.(2)Normal brain tissues and different grading glioma tissues were collected and tissue microarrays were prepared.Immunohistochemical staining for NKAP and Notch1 was performed.Expression changes in NKAP and Notch 1 were observed,and their expression relationships were analyzed.(3)Using luciferase reporter gene and ChIP assay to further explore the regulation of NKAP and Notch 1.(4)Rescue experiment:Plasmid transfected glioma cell U87MG to overexpress NKAP,add DAPT to block Notch signaling pathway,and explore the effect of blockade on the biological effect of U87MG overexpressing NKAP4.In vivo experiments in nude mice confirm that NKAP promotes the proliferation of glioma cells by regulating Notch 1The cells of U87MG NKAP ko and NKAP Con were collected and implanted subcutaneously in nude mice.Each group of cells was corresponding to 5 nude mice.The growth of the tumor was observed.When the implanted tumor grew under the skin,the tumor volume was measured regularly.Finally,the tumor was removed and the tumor weight was weighed and paraffin sections were made for immunofluorescence staining.In addition,nude mice were implanted intracranially with U87MG NKAP ko and NKAP Con.The survival of nude mice was counted,brain slices were taken,and HE staining was performed.Results1.Immunohistochemical staining and qRT-PCR assay showed that NKAP was localized in the nucleus of glioma cells.The expression of NKAP was little in normal brain tissue,while increased in gliomas.With the malignancy of glioma increased and the differentiation weakened,the expression of NKAP was gradually increased.(1)Compared with the control group,the OD(490)at 24h,48h,and 72h in the NKAP knockout group was significantly decreased,indicating that NKAP promoted the proliferation of glioma cells.EdU assay also showed that the NKAP ko group significantly reduced the number of positive cells,and the flow cytometric detection experiment clearly showed that the number of cells in the NKAP ko group was significantly reduced in the G1/S phase.(2)Transwell migration assay showed that the number of cells passing through the chamber was significantly decreased in the NKAP ko group.The Matrigel assay further demonstrated that NKAP promotes the migration and invasion of glioma cells.There was a significant reduction in the amount of cells injected into the bottom of the chamber membrane after NKAP knockout.3.(1)After the knockdown of NKAP in glioma cells,the expression of Notch1 in the mRNA level and the protein level was significantly reduced,no significant changes in other genes.(2)IHC staining showed that NKAP and Notch 1 were not expressed in normal brain tissue.With the malignant degree increased of glioma,the expression of both NKAP and Notch 1 were gradually increased.Statistical analysis showed a positive correlation between the expression of NKAP and Notch1 in tissues.(3)The luciferase reporter gene showed that Notch 1 transcriptional activity was decreased after U87MG NKAP knockout NKAP.ChIP experiments found that NKAP was significantly aggregated in the promoter of Notchl in U87MG.(4)The results of rescue experiments show that blockade of Notch signaling pathway can inhibit the biological effects of NKAP on glioma cells.4.in vivo experiments further confirmed that NKAP targeting Notch1 promotes glioma cell proliferation,and the growth rate of subcutaneous tumors.From the tumor growth curve,it can be seen that the expression of NKAP is reduced and the tumor growth rate is slowed down.Immunofluorescence results showed that the expressionof Notchl was also reduced in tumors with NKAP expression reduced.Survival:analysis showed that intracranial implanted glioma cells knocked out of NKAP glioma cells survive longer.Conclusions1.The expression of NKAP in gliomas is signficantly high.2.NKAP promotes the proliferation,migration and invasion of glioma cells.3.NKAP targets Notchl,activates the Notch signaling pathway,and promotes the developmentof gliomas.