The Expression Of MGMT In Glioma And Novel MicroRNA-4539 Suppresses MGMT Expression In Glioma Cells

Brain glioma is produced by the brain glial cell carcinogenesis,is the most common primary brain tumor,glioma incidence of 3 to 10/10 million,about intracranial tumors 46%,accounting for systemic malignancy 1%to 3%,with high mortality.To date,gliomas are mainly treated by surgical resection,followed by a combination of radiotherapy and temozolomide?TMZ?chemotherapy.TMZ is an alkylating agent that crosses the blood-brain barrier and induces apoptosis of tumor cells by generating methyl adducts that initiate futile cycling of the DNA mismatch repair pathway.But because of the high resistance of the tumor,clinical efficacy is unsatisfactory.Recent studies have shown that the cause of resistance to nitrosourea drugs is due to the presence of DNA repair enzyme 06-methylguanine DNA methyltransferase?MGMT?.MGMT reverses the DNA alkylation process by reversing the DNA alkylation process by transferring the alkyl from the 06 position of the guanine of the DNA to its cysteine residue,thereby recovering the proliferation of the tumor cells,and the glioma has an elevated MGMT activity chemical resistance to TMZ treatment.Some studies have shown that MGMT promoter methylation can silence the MGMT gene,thereby preventing DNA repair and enhancing the sensitivity of the tumor to alkylating agents.MicroRNA?miRNA?can combine the target gene 3'noncoding region,results in degradation of the target mRNA or inhibition of the translation process,and then regulate the expression of genes,participate in a series of biological behavior.Research Target:To analyze the expression of MGMT in glioma case specimens and to explore the relationship between MGMT expression level and glioma grade and size,to finding a miRNA that can affect MGMT expression will provide a new therapeutic target for drug resistance to glioma chemotherapy.Research method:A total of 47 patients with glioma tissue and previously underwent surgical from July 2015 to November 2015 were randomly selected from Qilu Hospital of Shandong University.All samples were identified as gliomas by an independent pathological examination.Each of the 47 glioma tissues were sliced into two parts.One tissue portion was prepared for histological and immunohistochemical staining,the other part is used to extract RNA.According to the classification of WHO central nervous system tumors?2007?,glioma specimens were classified as grade ?-? Grade ? and grade ? were classified as low malignant group,grade ? and grade IV were classified as high malignant group.The maximum diameter of the tumor was measured by fluidattenuated inversion recovery?FLAIR?.The expression of MGMT was quantified by H score in immunohistochemistry.SPSS 18.0 statistical software was used to analyze the data.The relationship between the positive rate of MGMT expression and the grade and size of glioma was tested by X2 test.The expression of MiRNA and mRNA were calculated by 2^-??ACT method.The t-test was used in the experimental group and the control group.P<0.05 had significant statistical difference.Searching the miR database?TargetScan database;http://www.targetscan.org/vert₆1/?to find a miRNA at the binding site of MGMT 3`UTR,and verified at the cellular level.The expression of MGMT and miRNA in glioma tissues was detected,clear the relationship between them.Construction of MGMT 3'UTR luciferase wild type and mutant vector,clear the relationship between them by double luciferase detection.At the cellular level,a relatively high cell line with MGMT expression was selected to be transfected with miR-4539 mimics,miR-4539 inhibitor,respectively to detected the MGMT expression.For functional verification,the expression of cck-8,apoptotic protein and flow cytometry were used to detect the cell proliferation and apoptosis of cells transfected with miRNA mimics and inhibitor.Results:1.The expression of MGMT in glioma tissues was not significantly correlated with the pathological grade and tumor size of gliomas.2.There was a negative correlation between the expression of MGMT and miRNA-4539 in glioma tissues;3.MiRNA-4539 can act on MGMT 3'UTR and inhibit its expression.4.In vitro,the expression of MGMT was decreased in cells transferred to miRNA-4539 mimics,the expression of MGMT was increased in cells transferred to miRNA-4539 inhibitor.5.In the in vitro cell level,when transfected into miRNA-4539 mimics,cell proliferation was slowed down and apoptosis increased,TMZ resistance decreased,and transferred into miRNA-4539 inhibitor,while TMZ resistance increased.Conclusion:MiRNA-4539 can improve the resistance of glioma cells to TMZ by inhibiting the expression of MGMT.