The Mechanism Of Histone Methyltransferase G9a On The Proliferation And Autophagy In Glioblastoma Cells

Gliomas are the most common intracranial primary tumors and account for about40%-50% of the total intracranial primary tumors.The cerebral hemisphere gliomas in this study account for approximately 51% of all gliomas.According to the classification of the tumor cells,astrocytomas are the most common gliomas,followed by oligodendrogliomas and oligoastrocytomas.The malignancy of astrocytomas can be divided into four grades: I,II,III,and IV.Grade I is astrocytomas,grade II is infiltrating cell tumors,and grade III is metaplastic tumors.Grade IV is glioblastoma,the most malignant glioma.Glioblastoma cell migration and invasion ability is very strong,coupled with tumor lesions in this critical area of the brain,patients with poor prognosis,clinical treatment is very difficult [1-3].The key oncogene known for glioblastoma is c-Myc.Genetic factors are generally believed to be one of the important causes of glioma occurance[4-5].In recent years,researchers have found that histone methyltransferase G9 a is highly expressed in many types of tumor cells.Abnormal histone methylation modification caused by this high expression can often cause changes in the target gene expression activity,thereby inducing tumorigenesis.Down-regulation of G9 a protein expression or using G9 a protein-specific small molecule inhibitors may lead to inhibition of the ability of tumor cells in proliferation,migration,invasion and autophagy [6-8].Although studies on the role of histone methyltransferase G9 a in glioblastoma have been reported,the mechanisms involved in the interaction between G9 a and the glioblastoma proto-oncogene c-Myc have hardly been studied.Therefore,the purpose of this study was to investigate the influence of histone methyltransferase G9 a on the proliferation,migration,invasion and autophagy of glioblastoma cells and its regulatorymechanism for glioblastoma oncogene c-Myc which can provide new ideas for the clinical treatment of glioma.The following are the main findings of this paper.1.Histone methyltransferase G9 a influences proliferation and tumor development of glioblastoma cellsWe detected the high expression of G9 a protein in glioblastoma cell lines by Western blot.Then we used the lentivirus-mediated shRNA interference technology and the specific inhibitor of G9 a protein BIX01294 to downregulate and inhibit the function of G9 a protein in LN-229 and U-87 MG cell lines,respectively.The results of CCK8 cell viability assay and Ki67 cell immunofluorescence assay showed that the proliferation of cells treated with G9 asi and BIX01294 were significantly inhibited,and cell flow experiments showed that the cell proliferation process was inhibited at the G2 phase.Western blot results showed that the expression levels of the proliferation-related key proteins were down-regulated in G9 asi cells and cells treated with BIX01294.The soft agar cloning experiment results showed that the in vitro colony formation ability of G9 asi cells and BIX01294 treated cells were significantly decreased.Finally,we established a tumor-bearing mouse model and found that G9 asi cells as well as cells treated with BIX01294 had reduced tumorigenicity in vivo.Analysis of the relationship between the expression of histone methyltransferase G9 a and the survival time of patients with glioblastoma in the online database of the R2-microarray analysis and visualization platform revealed that the higher the expression level of G9 a protein the worse the prognosis of the patients with glioblastoma.The above phenomenon shows that histone methyltransferase G9 a plays an active role in the proliferation and tumorigenesis of glioblastoma cells.2.Histone methyltransferase G9 a influences migration and invasion of glioblastoma cellsFirst,we performed a scratch test and the results showed that the migration ability of G9 asi cells and cells treated with BIX01294 was significantly reduced.Then we performed Transwell experiments.The results showed that G9 asi cells and cells treated with BIX01294 significantly reduced their migration and invasion ability.Finally,we detected the expression of key proteins involved in migration and invasion in G9 asi cells and cells treated with BIX01294 using Western blot technique.The results showed that the expression levels of these proteins were significantly downregulated.The abovephenomenon shows that histone methyltransferase G9 a plays an active role in the process of glioblastoma cell migration and invasion.3.Histone methyltransferase G9 a influences autophagy of glioblastoma cellsWe observed under the microscope that the morphology of G9 asi cells and cells treated with BIX01294 was significantly changed,that is,a large number of small vesicles and vacuoles clustered around the nucleus,which was morphologically very similar to the structure of autophagosomes.Followed by confocal results showed that the number of LC3B-positive plaques was significantly increased in G9 asi cells and cells treated with BIX01294.Finally,we detected the expression of key proteins associated with autophagy in G9 asi cells and cells treated with BIX01294 using Western blot technique,and the results showed that the expression levels of these proteins have changed significantly.The above phenomenon indicates that down-regulating the expression of histone methyltransferase G9 a or inhibiting its function can induce autophagy in glioblastoma cells.4.Histone methyltransferase G9 a modulates the transcription of the proto-oncogene c-Myc in glioblastomac-Myc has been shown to be an important proto-oncogene in gliomas.The transcriptome results predict that when the function of the histone methyltransferase G9 a protein is inhibited,the expression activity of the c-Myc gene in the cell is significantly inhibited.Then we detected the expression of c-Myc protein in G9 asi cells and BIX01294-treated cells using Western blot technique.We then over-expressed c-Myc in G9 asi cells and cells treated with BIX01294.It can be seen from the CCK8 cell viability test that the cell proliferation ability is restored before the c-Myc has not been overexpressed;the result of the soft agar cloning experiment shows that compared to the cell before the c-Myc was not overexpressed the ability of colony formation has also been restored.We then performed confocal experiments and the results showed that the number of LC3B-positive plaques in the cells was significantly reduced,that is,the autophagy of the cells was alleviated before the c-Myc was overexpressed.we established a tumor-bearing mouse model and found that the in vivo tumorigenicity of the cells was also restored before the c-Myc was overexpressed.Then we detected the expression of key proteins related to proliferation and autophagy in G9 asi cells and cells treated with BIX01294 using Western blot technique.Finally,based on theabove phenomena,we conducted ChIP-qPCR and Luciferase experiment.The results showed that the G9 a protein binds directly to the c-Myc promoter region,suggesting that histone methyltransferase G9 a directly regulates the transcriptional process of the glioblastoma proto-oncogene c-Myc,thereby affecting the overall occurrence of glioblastoma.In summary,histone methyltransferase G9 a plays an important role in the proliferation,tumorigenesis,migration,invasion,and autophagy of glioblastoma cells;histone methyltransferase G9 a can be directly controlled by the transcription process of the proto-oncogene c-Myc of the cytoblastoma affects the entire development process of glioblastoma.The results of this study provide a new strategy for the clinical treatment of glioblastoma,while enriching the theoretical basis of the disease.