The Mechanism Of Combined Inhibition Of BRD4 And HDAC3 To Induce Glioma Apoptosis Through GLI1/IL-6/STAT3 Signaling Pathway

Background:Malignant glioma is the most common intracranial tumor,which grows aggressively and high malignantly.The survival time of most patients can only reach12 to 15 months,and the 5-year survival rate is less than 5%^[1].As a highly heterogeneous tumor,malignant glioma has complex genetic and signal changes.At this stage,the main treatment method for malignant glioma is surgery,supplemented by chemotherapy,radiotherapy and targeted therapy.Even with targeted molecular targeted drugs,the clinical trial treatment effect is still not ideal^[2],which may be closely related to the existence of glioma stem cells.Therefore,from the perspective of glioma stem cells,exploring the underlying mechanism of the poor survival prognosis of patients with glioma is of great significance for the treatment of glioma,improving the prognosis and increasing the survival rate.In recent years,studies have found that there are abnormalities in epigenetic regulation in malignant gliomas.Epigenetic regulation can govern the dynamic transition of glioma cells between tumorigenic and non-tumorigenic states.Unlike genetic mutations,epigenetic changes are reversible^[3,4].Therefore,finding the key epigenetic sites of glioma and using epigenetic mechanisms to intervene to restore the normal state of the epigenetic modification of glioma cells is conducive to the development of new treatment strategies for glioma,providing a new direction for the treatment and prognosis of glioma patients.Histone deacetylase 3?HDAC3?is one of the class I members of the histone deacetylase?HDAC?family.It can catalyze the deacetylation of histones and other proteins and participate in a variety of transcriptional regulations of genes,which play an important role in cell development and tumors^[5,6].RGFP966 is a HDAC3 inhibitor discovered in recent years.Its target is HDAC3,and it has no effective inhibitory effect on other HDACs^[7].It has been reported in the literature that the HDAC3inhibitor,RGFP966,can inhibit the proliferation of skin T-cell lymphoma by inducing apoptosis^[8],and it can also inhibit tumor proliferation by promoting the differentiation of malignant hematological tumor cells^[9],suggesting that HDAC3expression is abnormal and HDAC3 is closely related to the occurrence and development of various tumors.In the preliminary work with sh RNA epigenetic library screening on glioma stem cells extracted from the spontaneous brain glioma model of transgenic mice?h GFAP-Cre⁺p53^L/^L Pten^L/⁺?^[10],we found that downregulation of HDAC3 can significantly inhibit the proliferation of mouse glioma stem cells?Glioma stem cells,GSC?.In the mouse model of glioma stem cell xenograft tumor,the HDAC3 inhibitor RGFP966?HDAC3i?could inhibit the growth of mouse glioma stem cell xenograft tumor,but it still showed a certain growth rate.There are documents confirming that the activation of leukemia inhibitory factor receptors limits the anti-tumor effects of histone deacetylase?HDAC?inhibitors in breast cancer.Therefore,it is speculated that those residual glioma stem cells that cannot be inhibited by HDAC3i may maintain abnormal self-renewal through the acetylation level of certain growth-promoting genes.BRD4 is a transcriptional and epigenetic regulator.Study found that BRD4,an important transcriptional elongation regulator,could recruit positive transcription elongation factor complex?P-TEFb?and caused activation of RNA polymerase II,thereby promoted the expressions of growth-promoting genes such as c-Myc.The acetylation of histones needs to be recognized by the"acetylation reader protein"before the final transcription result can be produced.BRD4 is a"reader protein"required in the transcription process mediated by histone acetylation.It has two tandem bromo domains?BD1,BD2?,and through the bromo domain,it recognizes and binds to the acetylated lysine site of histone end,and participates in the reading and regulation of epigenetic genes^[11].Among the four acetylation marker sites H3K14ac,H3K27ac,H3K122ac and H4K16ac,the combination of BRD4 and H3K27Ac sites is closely related to very high gene activity.The BRD4 selective inhibitor JQ1?BRD4i?can competitively bind to the bromodomain of the protein,thereby replacing the binding of BRD4 protein to the acetylated lysine on the chromatin and replacing BRD4,which can be used in NUT midline cancer,bone tumors and hematological tumors with good anti-tumor effects,and can significantly inhibit the STAT3 signaling pathway^[12,13].At the same time,previous work found that HDAC3i could induce the phosphorylation of STAT3 at tyrosine 705,and the activation of the STAT3 pathway is closely related to tumor growth.Therefore,if this activation could be inhibited,it is expected to enhance the inhibitory effect of HDAC3i on glioma stem cells,thereby further improving the therapeutic effect of HDAC3i on glioma.BRD4i could significantly inhibit the STAT3 signaling pathway in glioma stem cells.In conclusion,we speculated that the combination of the two may exhibit a strong anti-tumor effect.GLI1 is the terminal effector of Hh signaling pathway and transcription factor.GLI1 protein is an important transcription factor of the Hh signaling pathway,which can regulate downstream genes of the Hh pathway,and plays an important role in cell proliferation,differentiation,apoptosis and migration^[14,15].The literature has confirmed that the genomic footprint of BRD4 in neuroblastoma overlaps the part occupied by GLI1,and BRD4 can directly regulate the transcriptional activity of Gli?Gli1 and Gli2?^[16,17].As well,GLI1 could bind to the IL-6 promoter and regulate the activity and expression of IL-6 to maintain activated STAT3^[18].On this basis,this study was to compare the effects of HDAC3i and BRD4i in combination with HDAC3i on glioma cell proliferation,apoptosis and STAT3signaling pathway,and observe the changes of glioma under the administration of BRD4i combined with HDAC3i,further verify the synergistic effects of BRD4i and HDAC3i in the treatment of glioma,and analysis of the way of BRD4i and HDAC3i regulating STAT3 signaling pathway,reveal the mechanism of BRD4i and HDAC3i synergistic inhibition of glioma to find new therapeutic targets for glioma.Objective:1.Take GSCs as the experimental object,combined with BRD4,to explore the effect of BRD4i combined with HDAC3i on the growth of glioma stem cells;2.Explore the effect and mechanism of action of BRD4i combined with HDAC3i on glioma stem cells.Method:1.The effect of combined inhibition of BRD4 and HDAC3 on glioma stem cells.In vitro experiments:Experimental group:negative control group,BRD4i or si BRD4 group,HDAC3i group,BRD4i and HDAC3i group.The effect of CCK8 on the viability of GSCs cells.The effect of cell growth count and colony formation assay on the value-added.The ability of neurospheres to detect cell self-renewal.The change of apoptosis and cell cycle by flow cytometry;TUNEL staining Apoptosis ability;q RT-PCR was used to detect the changes of apoptosis and cycle-related genes and differentiation markers;immunofluorescence was used to detect the expressions of relevant differentiation markers.In vivo experiment:Experimental group:negative control group,BRD4i group,HDAC3i group,BRD4i and HDAC3i group.During the administration of the drug,the tumor diameter was observed and measured every other day,and the body weight of the mice was weighed,and the mice were sacrificed for statistical analysis;immunohistochemical staining was used to observe the effects of markers on glioma stem cells-related proliferation and differentiation;TUNEL staining was used to detect apoptosis;q RT-PCR was used to detect apoptosis and cycle-related genes and expressions of differentiation markers;Western-blot analysis of STAT3 and expressions of downstream gene proteins Bcl-2 and Mcl-1.2.Combined inhibition of BRD4 and HDAC3 to inhibit the growth of malignant glioma stem cells.Different concentrations of HDAC3i solution were applied to CSC2078 cells.The activity of HDAC3 was verified by ELISA kit.The effect of HDAC3i on CSC2078cells was determined by CCK8.The changes of STAT3 pathway and downstream gene protein levels were detected by Western-blot.Using sh STAT3 and HDAC3i to act on CSC2078 cells,Western-blot analysis of STAT3 and downstream gene Bcl-2,Mcl-1 protein levels;cell growth count assay to analyze cell proliferation ability;neurosphere test to detect cell self-renewal ability.Different concentrations of BRD4i solution were applied to CSC2078 cells,and the expressions levels of STAT3 and p-STAT3?Tyr705?were detected by western-blot assay.RNA-seq sequencing was used to analyze the key molecules of BRD4i combined with HDAC3i in inhibiting the growth of glioma stem cells;Western-blot was used to verify the effect on STAT3 signaling pathway;Ch IP was used to analyze the acetylation of GLI1 gene H3K27 by HDAC3i and the expression of BRD4 at GLI1promoter;IL-6 secretion by ELISA;Ch IP analysis of GLI1 regulation of IL-6;Western-blot analysis of IL-6 and STAT3 pathway changes.Result:1.Combined inhibition of BRD4 and HDAC3 can effectively inhibit the growth of glioma stem cells.In vitro experiments:The results of CCK8 showed that BRD4i combined with HDAC3i group had the most obvious inhibitory effect on the cell viability of GSCs,and showed significant synergistic effect,compared with the negative control group,BRD4i or si BRD4 group and HDAC3i group;cell growth count results showed that the cells of BRD4i or si BRD4 combined with HDAC3i group were significantly inhibited and the number was the least,compared with the negative control group,BRD4i or si BRD4 group and HDAC3i group;the colony formation experiment confirmed that the number of clones formed was the least,compared with the negative control group,BRD4i group and HDAC3i group;the results of neurosphere experiments showed that the cells in BRD4i or si BRD4 combined with HDAC3i had the worst self-renewal ability compared with the negative control group,BRD4i or si BRD4 group and HDAC3i group;flow cytometry results showed that compared with the negative control group,BRD4i or si BRD4 group and HDAC3i group,BRD4i or si BRD4 combined with HDAC3i group had the highest number of apoptosis cells,and most of the cells had G1 phase cell arrest;TUNEL staining showed that BRD4i combined with HDAC3i group had the highest number of green fluorescence cells and strong fluorescence intensity,compared with the negative control group,BRD4i group and HDAC3i group;q RT-PCR results showed that the expressions of apoptosis and cycle-related genes and differentiation markers in BRD4i combined with HDAC3i group changed significantly,compared with the negative control group,BRD4i group and HDAC3i group;immunofluorescence results showed the expressions of the relevant differentiation markers in the BRD4i combined with the HDAC3i group was the most obvious,compared with the negative control group,BRD4i group and HDAC3i group.In vivo experiment:The tumor size and mouse body weight were analyzed.The results showed that the BRD4i combined with HDAC3i group had the smallest tumor volume,while the four groups of mice did not change the weight,Compared with the negative control group,BRD4i group and HDAC3i group;histochemical staining showed that the markers of proliferation and differentiation in BRD4i combined with HDAC3i group were the most significant compared with the negative control group,BRD4i group and HDAC3i group.The results of TUNEL staining showed that the apoptosis ability of BRD4i combined with HDAC3i group was significantly increased compared with the negative control group,BRD4i group and HDAC3i group;q RT-PCR results showed the expressions of apoptosis and cycle-related genes and differentiation markers in BRD4i combined with HDAC3i group were changed mostly compared with the negative control group,BRD4i group and HDAC3i group;the results of Western-blot showed that the expressions of STAT3 and downstream gene proteins Bcl-2 and Mcl-1 in BRD4i combined with HDAC3i group were significantly inhibited compared with the negative control group,BRD4i group and HDAC3i group.2.Combined inhibition of BRD4 and HDAC3 induces glioma stem cell cycle arrest and apoptosis through GLI1/IL-6/STAT3 signaling pathway.ELISA kit confirmed that HDAC3i could significantly inhibit the activity of HDAC3;CCK8 results showed that HDAC3i had a certain inhibitory effect on the viability of CSC2078 cells,and the inhibitory effect at the maximum concentration is less than 60%;Western-blot results showed that HDAC3i could induce STAT3 at 705phosphorylation and increased levels of Bcl-2 and Mcl-1 proteins in downstream,and sh STAT3 significantly inhibited the activation of STAT3 induced by HDAC3i and the expressions of Bcl-2 and Mcl-1.The results of cell growth counting showed that compared with the negative control group and the HDAC3i group,the number of cells in the sh STAT3 combined with HDAC3i group was significantly reduced.The results of the neurosphere experiment confirmed that the ability of the sh STAT3 combined with the HDAC3i group was significantly inhibited,compared with the negative control group and the HDAC3i group;Western-blot results showed that BRD4i could cause different degrees inhibition of p-STAT3?Tyr705?protein expression in CSC2078 cells.RNA-seq data analysis showed that compared with the control group,there were1294 overlapping genes between GSCs treated with HDAC3i and GSCs treated with BRD4i combined with HDAC3i.Among the 1294 genes,35 genes were up-regulated after being induced by HDAC3i,but after the addition of BRD4i,their expression was significantly suppressed.As shown in Figure 5A,relative transcription level=log 2?fold change of each gene?.In this case,the synergy index of each gene is calculated based on the relative transcription level?|BRD4i&HDAC3i|-|BRD4i|+HDAC3i?.For example,the relative transcription levels of Gm20661 are-3.829?BRD4i group?,2.205?HDAC3i group?and-1.167?BRD4i&HDAC3i group?,so the synergy index of Gm20661 is-0.457?|-1.167|-|-3.829|+2.205?.According to this calculation method,among these 35 genes,GLI1 has the strongest synergy index.Therefore,it is speculated that GLI1 is the key molecule in the process of BRD4i combined with HDAC3i to inhibit the growth of glioma stem cells;Western-blot confirmed that combined inhibition of BRD4 and HDAC3 can significantly inhibit the expression of GLI1,while down-regulation of GLI1 can inhibit the STAT3 signaling pathway,and si GLI1 combined with HDAC3i can Significantly inhibit the STAT3 signaling pathway and the expression of Bcl-2 and Mcl-1;Ch IP-q PCR results show that HDAC3i can enhance the acetylation level of H3K27 at the promoter of GLI1 gene and recruit more BRD4 from the promoter of GLI1 gene to participate in transcription;QRT-PCR and Western-blot confirmed that the down-regulation of GLI1 can inhibit the m RNA level and protein level of IL-6.At the same time,ELISA results show that si GLI1 can reduce the secretion of IL-6;Ch IP-q PCR results show that GLI1 can start with IL-6 Binding directly regulates the transcription of IL-6;Western-blot results show that si IL-6 can inhibit the STAT3 pathway,and this inhibition can be restored by exogenous IL-6 at the same time.Conclusion:1.BRD4i combined with HDACi synergistically inhibits the proliferation and and self-renewal of glioma stem cells,induces cycle arrest and apoptosis of glioma stem cells,and induces them to differentiate into mature astrocytes.2.HDACi can enhance the acetylation of GLI1 genomic histone H3K27,activate the IL-6/STAT3 signaling pathway,and limit the inhibitory effect of HDACi on glioma stem cells;combined with BRD4i,it inhibits the transcription of GLI1 and further inhibits IL-6/STAT3 pathway to play a synergistic anti-tumor effect.