Research On H3K27me3 Expression In Glioma And Its Effect On Temozolomide Sensitivity

Objective:Histone H3 27-site lysine methylation（H3K27me3）is an important epigenetic modification that has been found to be associated with chemotherapy resistance.In this study,the relationship between H3K27me3 expression and temozolomide sensitivity was analyzed by detecting the expression of H3K27me3 in glioma patients and cell lines and intervening the expression of H3K27me3 in cell lines to change temozolomide sensitivity,aiming to provide an important reference for reversing temozolomide resistance for glioma patients in clinical practice.Methods:1.Clinical research:Surgical pathological sections of 20patients with high-grade glioma were collected,the expression of H3K27me3 was detected by immunohistochemical method and its clinical significance was analyzed.2.Cell and molecular experiments:Firstly,the sensitivity difference between glioma cell line U87 and temozolomide-resistant cell line U87TR to temozolomide were detected by CCK-8,the expression of H3K27me3 and lysine demethylase 6B（KDM6B）in U87 and U87TR were detected by q RT-PCR and Western blotting.Secondly,the expression of H3K27me3 and KDM6B in glioma cells were detected after treatment with different concentrations temozolomide by q RT-PCR and Western blotting.Thirdly,CCK-8 and Transwell assays were used to detect the effects of H3K27me3 demethylase inhibitor GSK-J4 at different concentrations combined with temozolomide on the proliferation and migration of U87TR cells.Finally,q RT-PCR and Western blotting were employed to detect the expression of H3K27me3 and KDM6B after combined treatment with GSK-J4 and temozolomide 3.Statistical analysis:SPSS 26.0 and Graphpad prism 9.4.0 were used for statistical analysis.The effect of different grades on the expression level of H3K27me3 was explored by Fisher’s exact probability method.Kaplan-Meier curve and Cox regression model were used to explore the relationship between H3K27me3 expression and the prognosis of glioma.Spearman correlation analysis was used to explore the correlation between the expression of H3K27me3 and KDM6B.Cellular and molecular experimental results were compared between groups using single factor analysis of variance.P<0.05 was regarded as a statistically significant difference.Results:1.As for clinical research,immunohistochemical staining and scoring was performed on 20 cases of high-grade glioma sections.The high expression rate of H3K27me3 was higher in grade 3 glioma than in grade 4glioma,suggesting significant difference（P<0.01）.（P<0.01）.The Kaplan-Meier curve indicated that the higher the H3K27me3 expression,the longer the disease free survival（DFS）and overall survival（OS）of glioma patients（P<0.05）.Univariate Cox regression analysis showed that the higher the H3K27me3 expression,the longer the DFS of patients with high-grade glioma,showing significant difference.Spearman correlation analysis indicated that the expression of H3K27me3 was negatively correlated with H3K27 demethylase KDM6B（r=-0.74,P<0.01）.2.Results of cell and molecular experiments indicated that the half-inhibitory concentrations of U87 and U87TR cells to temozolomide were 408μmol L^-1 and 708μmol L^-1,respectively,and there was a significant statistical difference（P<0.01）.The expression of KDM6B was significantly higher in U87TR than in U87（P<0.01）,while the level of H3K27me3 was significantly lower（P<0.01）.Temozolomide treatment could increase the expression of KDM6B in two glioma cell lines（P<0.05）,and significantly reduce the expression of H3K27me3（P<0.01）.GSK-J4 treatment can significantly increase the H3K27me3 in U87TR cells expression（P<0.01）.GSK-J4 could increase the inhibitory effect of temozolomide on the proliferation and migration of U87TR（P<0.01）.Conclusions:1.The H3K27me3 expression is negatively correlated with the malignancy degree of glioma,and high expression of H3K27me3 indicates a better prognosis.2.The decreased expression of H3K27me3 in temozolomide-resistant glioma cells may be one of the causes of drug resistance.3.Histone H3K27 demethylase inhibitor GSK-J4 can increase the expression of H3K27me3,thereby increasing the sensitivity of drug-resistant glioma cells to temozolomide.4.Increasing the expression of H3K27me3 is expected to be one of the strategies to reverse the temozolomide resistance in the treatment of glioma.