The Role And Mechanism Of Histone Demethylase JMJD3 In Invasion,Proliferation,and Senescence Of Glioma Cells

Glioma is one of the most common malignant brain tumors,accounting for about 40% of the total brain tumors and 80% of all malignant brain tumors.Glioma patients were treated mainly with surgery,radiotherapy and chemotherapy,most of which has a poor prognosis.So,it is of great significance to explore new therapeutic strategies for glioma.A series of research advances on glioma mechanism provide numerous therapeutic targets.Epigenetics is the study of heritable changes in gene function that do not involve changes in the DNA sequence,including DNA methylation,RNA interference,and posttranslational modification(PTM)of histone.Histones are the chief protein components of chromatin.On the one hand,histones act as structural scaffolds to maintain chromosomal integrity.On the other hand,they play an important regulatory role in gene expression.There are a wide range of PTMs of histones,such as acetylation,methylation,ubiquitination and phosphorylation,etc.These modifications ultimately affect specific gene expression through transcriptional activation or repression.Histone methylation is a common posttranslational modification,mainly focused on the lysine and arginine residues of histone H3 and H4.The specific residues of histone can be modified by monomethylation,dimethylation,and trimethylation,and most common residues include histone H3 lysine 4(H3K4),lysine 9(H3K9),ly27(H3K27),and lysine 36(H3K36).JMJD3(Jumonji domain-containing protein 3)is an H3K27 demethylase which mainly catalyzes the demethylation of methylated H3K27(H3K27me2/3).Because H3K27me2/3 is a transcriptional repression marker,JMJD3 has the activity of transcriptional activation.It has been found that there were abnormal expressions or activities of JMJD3 in many cancers,including lung cancer,prostate cancer,and renal cancer.But the role and function of JMJD3 on gliomas have been less studied.Therefore,this study investigated the role and mechanism of JMJD3 in the gliomas development,so as to expand the understanding of glioma.This study also explores potential applications of JMJD3 in order to provide a new strategy for the treatment and prevention of gliomas.Part 1 The role and mechanism of histone demethylase JMJD3 in invasion of gliomaObjective: To explore the role and potential mechanism of histone demethylase JMJD3 in the glioma invasion in order to find the target for diagnosis,treatment and prevention of glioma.Methods: Two human glioma cell lines,U87 and U251,were purchased from the Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences.Both cells were cultured in DMEM medium containing 10% fetal bovine serum and maintained at 37°C with 5% CO2.The plasmid of CRISPRi or CRISPRa for JMJD3 was transfected into two glioma cells using Lipo3000,and the effect of inhibition or overexpression was evaluated with qRT-PCR and Western blotting.A scratch assay was performed to determine the migration ability of both two glioma cells U87 and U251 after JMJD3 overexpression or inhibition.A transwell assay was performed to assess the migration abilities of U87 and U251 in JMJD3 activation of suppression.The expression of key gene of epithelial-mesenchymal transition(EMT),SNAI1,was measured with qRT-PCR and Western blotting.Chromatin immunoprecipitation(ChIP)technique was performed to identify the binding ability of JMJD3 to the regulatory of SNAI1 gene region and the status of H3K27me3.Results:1.The overexpression of histone demethylase JMJD3 in glioma tissuesOncomine database analysis showed that the expression of JMJD3 was increased significantly in glioblastoma(542 cases)compared with normal brain tissue(10 cases)(P<0.05).It was initially demonstrated that JMJD3 was closely associated with gliomas.2.CRISPRi and CRSIPRa are effective methods in inhibiting or increasing the expression of histone demethylase JMJD3 in glioma cells.The qRT-PCR,Western blotting and immunofluorescence results showed that CRISPRi-JMJD3 can significantly inhibit the expression of JMJD in glioma cells(P<0.05)and increase histone H3K27me3 content.CRISPRa-JMJD3 can significantly upregulate the expression of JMJD3 in cells(P<0.05),and reduce histone H3K27me3 level.These results demonstrated that the CRISPRi or CRSIPRa is an effective and rapid strategy to modify the expression of endogenous genes in cells.3.Histone demethylases JMJD3 can affect cell migration and invasionThe results of the scratch assay showed that down-regulation of JMJD3 expression significantly inhibited the migration of both glioma cells,while overexpression of JMJD3 significantly increased cell migration(P<0.05).Transwell assay further showed that the expression of JMJD3 was positively correlated with cell invasion(P<0.05).4.The histone demethylase JMJD3 can bind to the regulatory region of SNAI1 gene and reduce histone H3K27me3 levels,thereby upregulating SNAI1 expression.CRISPRi/a experiments showed that JMJD3 expression was also consistent with the expression of the key gene of EMT,SNAI1.The ChIP experiments further indicated that JMJD3 can bind to the regulatory region of SNAI1 and reduce H3K27me3 content due to its H3K27 demethylase activity.Therefore,these results illustrated that JMJD3 promotes cell migration and invasion partly through direct regulation of SNAI1 expression.Summary: The histone demethylase JMJD3 is an important promoting factor of glioma invasion and migration,and can directly affect the expression of EMT-associated factor SNAI1.This finding has important implications for the diagnosis and treatment of gliomas.Part 2 The effect of histone demethylase JMJD3 inhibitor GSK-J4 on glioma cellsObjective: To investigate the effects of histone demethylase JMJD3 inhibitor GSK-J4 on the proliferation,apoptosis and migration of glioma cells.Methods: Glioma cells U87,U251 and endothelial cells hCMEC were cultured in DMEM medium containing 10% fetal bovine serum and maintained at 37°C with 5% CO2.The expression of JMJD3 in three cells was detected by qRT-PCR and Western blotting.The levels of JMJD3 and H3K27me3 were determind using Western blotting after three glioma cells were treated with GSK-J4,which were used to determine the physiological activity of GSK-J4.The effect of GSK-J4 on cell proliferation was detected by CCK-8 assay after three cells were treated with different concentration or in different time.The function of GSK-J4 on cell apoptosis was measured with flow cytometry assay.Results:1.Overexpression of JMJD3 in glioma cellsThe results of qRT-PCR and Western blotting showed that the expression of JMJD3 at mRNA and protein levels was significantly higher in two glioma cells than that in endothelial cell(P<0.05).At the same time,the content of H3K27me3 in glioma cells was reduced.All these results suggested that JMJD3 overexpression and Hypomethylation of H3K27 may be associated with glioma cell development and may serve as a therapeutic target.2.Histone demethylase inhibitor GSK-J4 increases H3K27me3 contentWestern blotting results showed that GSK-J4 significantly increased H3K27me3 content in U87 and U251 glioma cells,but had no significant effect on endothelial cells,indicating that GSK-J4 has a histone H3K27me3 demethylase inhibitory activity.3.GSK-J4 inhibits the cell proliferation of glioma cellsThe results of CCK-8 indicated that GSK-J4 had significant inhibitory effect on cell proliferation of U87 and U251,and the inhibition was dose-dependent(P<0.05)and time-dependent(P<0.05).There is not obvious inhibition on endothelial cell proliferation of GSK-J4.This result showed that GSK-J4 is a glioma cell-specific proliferation inhibitor.4.GSK-J4 induces apoptosis of glioma cellsThe results of flow cytometry showed that GSK-J4 significantly induced the apoptosis of U87 and U251 glioma cells(P<0.05),but had no significant effect on the apoptosis of hCMEC cells.This result illustrates that GSK-J4 is a glioma-specific apoptosis inducer with relatively high safety.5.GSK-J4 inhibits glioma cell migrationThe cell scratch assay showed that GSK-J4 can reduce the migration ability of U87 and U251(P<0.05 and P<0.01),indicating that GSK-J4 also has important potential in the inhibition of glioma metastasis.Summary: GSK-J4,a specific inhibitor of histone H3K27 demethylase JMJD3,can selectively inhibit the proliferation and migration of glioma cells and promote apoptosis.This finding provides a new strategy for the treatment of glioma.Part 3 The role and mechanism of histone demethylase JMJD3 in 1,25-dihydroxyvitamin D3 induced senescence of glioma cellsObjective: To study the role of JMJD3 on the senescence of glioma cells induced by 1,25-dihydroxyvitamin D3 and its potential mechanism,so as to provide an important strategy for the prevention and adjuvant treatment of glioma.Methods: The SA β-gal colorimetric method was used to study the effect of 1,25-dihydroxyvitamin D3 on senescence of glioma cells U87 and U251.The mRNA expressions of histone demethylase JMJD3,vitamin D receptor(VDR)and senescence marker INK4 A were measured with qRT-PCR.These protein contents of JMJD3,VDR,INK4 A and H3K27me3 were determined with Western blotting and immunofluorescence.Chromatin immunoprecipitation(ChIP)was used to detect the binding of JMJD3 and VDR in regulatory region of INK4 A gene and the content of H3K27me3.Results:1.1,25-dihydroxyvitamin D3 increases the senescence of glioma cells U87 and U251The SA β-gal colorimetric assay showed that 1,25-dihydroxyvitamin D3 treatment significantly increased the senescence of glioma cells U87 and U251 cells(P<0.05),thereby achieving the purpose of inhibiting cell proliferation.So,1,25-dihydroxyvitamin D3 also has a certain inhibitory effect on gliomas.2.1,25-dihydroxyvitamin D3 increases expression of histone demethylase JMJD3 and cell senescence marker INK4AqRT-PCR,Western blotting and immunofluorescence showed that 1,25-dihydroxyvitamin D3 increased the mRNA and protein contents of histone demethylase JMJD3 in glioma cells(P<0.05).The expression of INK4 A was also induced by 1,25-dihydroxyvitamin D3(P<0.05).All these results demonstrated that both JMJD3 and INK4 A may be involved in 1,25-dihydroxyvitamin D3-induced cell senescence.3.Inhibition of JMJD3 attenuates 1,25-dihydroxyvitamin D3-induced cell senescenceDown-regulation of JMJD3 using RNA interference can significantly inhibit 1,25-dihydroxyvitamin D3-induced cell senescence and the up-regulation of INK4A(P<0.05).It demonstrated that JMJD3 mediates 1,25-dihydroxyvitamin D3 induced cell senescence.4.JMJD3 directly regulates INK4 A expression induced by 1,25-dihydroxyvitamin D3The results of ChIP experiment showed that 1,25-dihydroxyvitamin D3 can significantly increase the binding of JMJD3 and VDR to the promoter of INK4 A and reduce histone H3K27me3 level(P<0.05).This result indicated that the JMJD3 catalyzed demethylation of H3K27me3 in INK4 A promoter is one of the mechanisms by which 1,25-dihydroxyvitamin D3 induces INK4 A expression and thus promotes senescence.Summary: 1,25-dihydroxyvitamin D3 can promote the senescence of glioma cells,the mechanism of which is to increase the expression of JMJD3.Then JMJD3 can further combine with the vitamin D3 receptor(VDR)to binding the promoter of INK4 A to increase the expression of INK4 A by reducing the H3K27me3 level.This finding is of great value for glioma prevention and adjuvant therapy.Conclusion: The expression of histone demethylase JMJD3 is higher in glioma than that in paracancerous tissues.Overexpression or downregulation of JMJD3 can enhance or reduce the migration of glioma cells.JMJD3 can upregulate the expression of SNAI1 through demethylaion of H3K27me3 in the promoter of SNAI1 gene.GSK-J4,a specific inhibitor of JMJD3,can decrease the proliferation and migration of glioma cells and promote apoptosis.But,there is no significant effect of GSK-J4 on endothelial cells.1,25-dihydroxyvitamin D3 can promote the senescence of glioma cells.1,25-dihydroxyvitamin D3 can increase the expression of JMJD3,which can further increase the expression of INK4 A by catalyzing the demethylation of H3K27me3 in the promoter of INK4 A gene.Increases cellular senescence.These results are of great significance for the diagnosis and treatment of gliomas.