The Bromodomain Inhibitor JQ1 Induced Cell Cycle Arrest And Apoptosis Of Glioma Stem Cells Through VEGF/PI3K/AKT Signaling Pathway

Background:Glioma is the most common malignant intracranial tumor with the highest mortality.There are many treatments of malignant glioma including surgery,radiotherapy and chemotherapy,which are less effective in prognosis and survival period in most patients especially in glioblastoma multiform(GBM)patients whose five-year survival period rate is less than 5%.Therefore,it is urgent to explore more effective methods to treat glioma.In recent years,many researches have reported Brd4 as a therapeutic target in many cancers,and BET bromodomain inhibitor JQ1 plays an essential anti-cancer role in many kinds of cancers.However,there are few researches on the effects and molecular mechanism in regulating GBM process by Brd4 and JQ1.In addition,most researches about GBM mainly use glioma cell lines like U87 and U251 as experimental research objects rather than glioma stem cells(GSCs).With the continuous deepening of research on glioma,glioma stem cells are increasingly considered as the driving force for the development of glioma and the main reason for the resistance of glioma to therapeutic drugs.Glioma stem cells have strong self-renewal ability,multidirectional differentiation potential and strong tumorigenic ability,which makes GBM have tumor heterogeneity.Therefore,in this study,we used glioma stem cells for experimental research,which makes our research results more valuable and convincing.Objective:In this study,we chose GSCs as experimental object,using the BET bromine JQ1 structure domain inhibitors or specific targeted Brd4 si RNA to inhibit protein expression and function.And the effects of JQ1 or si Brd4 on the viability,proliferation,self-renewal,cell cycle and apoptosis in GSCs were examined in vivo and in vitro,and the mechanism of their effects was discussed.At the same time,the effect of JQ1 combined with temozolomide(TMZ)in GBM was also discussed.Method:In vitro,the experiment groups were divided into JQ1 group and si Brd4 group.JQ1 group was divided into control group and JQ1 group with different concentrations;si Brd4 group was divided into control group,NC group and si Brd4 group(si Brd4-475,si Brd4-1648,si Brd4-3820).(1)Brd4 is a potential therapeutic target for glioma.The number of copies of Brd4 in GBM was analyzed by TCGA database,and the expression of Brd4 in human glioma tissues at different levels was detected by immunohistochemical staining.(2)The effects of JQ1 on cell viability,proliferation,and self-renewal ability of GSCs.CCK8 assay was used to detect the effect of JQ1 on cell viability of GSCs,and the dependence of GSCs on the concentration and time of JQ1.Cell growth curve and soft agar colony formation assays were used to detect cell proliferation of CSC2078 treatment by JQ1.The effect of JQ1 on self-renewal ability was detected by the neural sphere formation assays.(3)The effects of JQ1 or si Brd4 on cell cycle and apoptosis of GSCs.The PI staining flow cytometry was used to detect the effects of JQ1 or si Brd4 on cell cycle of GSCs.The Annexin V/PI staining flow cytometry and Tunnel staining were used to detect the effect of JQ1 or si Brd4 on apoptosis of GSCs.(4)The effect of JQ1 or si Brd4 on the mechanism and signaling pathway of GSCs.The genome-wide m RNA expression level of GSCs was detected by RNA-seq.The effect of differentially expressed genes of GSCs treated by JQ1 and the mechanism of JQ1 effect on GSCs and the enriched signal pathways were analyzed by GO analysis and KEGG(Kyoto encyclopedia of Genes and Genomes).The m RNA and protein expression levels of key genes about the signaling pathway were detected by q-PCR and Western blotting.(5)The therapeutic effect of JQ1 on GSCs xenograft mouse model.1× 107CSC2078 were subcutaneously injected into BALB/c nude mice to construct the xenograft mouse model.After 2 days,nude mice of JQ1-treated group were administered intraperitoneally with JQ1(50 mg/kg/day).The volume of tumor tissues was analyzed statistically.The effect of JQ1 on apoptosis of GSCs was detected by Tunel fluorescence staining in mouse tumor tissue sections.HE staining and immunohistochemical staining were used to detect the effects of tumor cell morphology and the protein expression level of various markers such as proliferation,cycle and apoptosis.HE staining was used to detect whether JQ1 had toxicity in mice.The expression of important proteins in the VEGF/PI3K/AKT signal pathway was detected by Western blotting.(6)The effects of JQ1 combined with TMZ.Western blotting was used to detect the m RNA and protein expression level of MGMT in CSC2078 transfected with si Brd4.CCK8 assay was used to detect the effects of JQ1,TMZ and JQ1 combined with TMZ on cell viability of CSC2078.The Compu Syn software was used to calculate the CI index of JQ1 combined with TMZ.The PI staining flow cytometry was used to detect the effect of JQ1,TMZ,JQ1 combined with TMZ on cell cycle of CSC2078.The Annexin V/PI staining flow cytometry was used to detect the effects of JQ1,TMZ and JQ1 combined with TMZ on apoptosis of CSC2078.Result:(1)TCGA database analysis showed that the copy number of Brd4 in GBM was significantly higher than astrocytoma.Immunohistochemical staining results of human glioma tissue samples of different pathological grades showed that Brd4 was highly expressed in human glioma tissue samples of grade III and IV.(2)JQ1 or si Brd4 inhibit GSCs activity,cell proliferation and self-renewal,and promote cell cycle arrest and apoptosis of GSCs.(3)The results of RNA-seq suggested that the anti-tumor effect of JQ1 or si Brd4 above on GSCs was closely related to the PI3K/AKT signaling pathway and significantly inhibited the expression of vascular endothelial growth factor(VEGF)in PI3K/AKT signaling pathway.JQ1 combined with Linifanib,a tyrosine kinase inhibitor of VEGFR2,further demonstrated that JQ1 exerted anti-tumor effect on GSCs through the VEGF/PI3K/AKT signaling pathway.After treatment with GSCs by JQ1,the expressions of cyclical-related target genes c-Myc,Cyclin D1,Rb and E2F1 in the downstream of VEGF/PI3K/AKT signaling pathway were decreased,while the expressions of P21 and P27 were increased.After the treatment of GSCs with JQ1,the expression of pro-apoptotic genes was increased and the expression of inhibiting apoptotic genes were decreased in the VEGF/PI3K/AKT signaling pathway downstream.Moreover,JQ1 or si Brd4 promoted the expression of ?H2AX,and increased DNA damage further promoted the apoptosis of GSCs.(4)In vivo experiments showed that JQ1 could effectively inhibit the growth of tumor volume.The expression of c-Myc,PCNA,BCL-2 and MMP in the tumor of the JQ1 treatment group were decreased,while the expression of BAX protein was increased.The expression of key genes about PI3K/AKT signaling pathway was consistent with the results of in vitro cell experiments.(5)Silencing Brd4 effectively reduced the m RNA and protein expression levels of MGMT.Compared with TMZ-treatment group and JQ1-treatment group,TMZ and JQ1 combined group had a more significant inhibitory effect on cell viability of GSCs.Conclusion:(1)In vivo and vitro experiments,inhibition of Brd4 expression or function can promote GSCs cycle arrest and apoptosis through the VEGF/PI3K/AKT signaling pathway.(2)JQ1 reduces MGMT level by affecting the function of Brd4,thus enhancing the anti-tumor effect of TMZ.(3)Brd4 is an effective therapeutic target for GBM.