| Background:Malignant glioma is the most common intracranial malignant tumor with high mortality rate.It has highly invasive,highly drug resistant and high recurrence rate.Glioma stem cell(GSC)is one of the important reasons for inducing the occurrence of glioma,causing the resistance of glioma chemoradiotherapy and tumor recurrence.Conventional chemoradiotherapy is designed to kill rapidly proliferating tumour cells.Cancer stem cells,on the other hand,are relatively static and could transport drugs from the cells and reducing the drug's effect.At the same time,cancer stem cells can reduce the damage caused by radiotherapy by maintaining low levels of reactive oxygen.Therefore,traditional chemoradiotherapy is not effective for cancer stem cells and can cause tumor recurrence after treatment.Therefore,targeting GSCs may be a new strategy for the treatment of malignant glioma.At present,the main strategies for cancer stem cells are to change their self-renewal related signaling pathways and change their microenvironmental pathways,but there are still few studies on glioma stem cells.Abnormal epigenetic modifications play an important role in the maintenance of tumor phenotypes.For the pathogenesis of GBM,glioma stem cells may have abnormal epigenetic modification to maintain the stem cell state of undifferentiated or incomplete differentiation.But unlike gene mutations,epigenetic modifications are often reversible.If the glioma stem cells are induced to differentiate into mature glial cells,it is expected to reduce the malignant degree of the tumor and inhibit the chemoradiotherapy resistance of the tumor stem cells.Histone acetyltransferase(HATs)and histone deacetylase(HDACs)are often imbalanced in malignant glioma.The overexpression of histone deacetylase 3(HDAC3)is closely related to glioma.The overexpression of HDAC3 can increase the level of histone deacetylation and lead to the decreased expression of some tumor suppressor genes such as p53 and p21,thus leading to the occurrence of tumor.Histone deacetylase inhibitors can inhibit the effect of histone deacetylase,and thus play a role in the treatment of tumors.At the same time,epigenetic therapy can be used to intervene at the post-translational modification level to achieve more precise effects.Studies have shown that inhibition of HDAC3 can promote tumor cell apoptosis and cell cycle arrest,but the effect on glioma stem cells is not clear.In the previous work,the screening of epigenetic chromatin regulators of glioma stem cells showed that down-regulation of HDAC3 gene could not only promote proliferation of GSCs,but also significantly promote differentiation.Therefore,it is speculated that inhibition of HDAC3 can induce differentiation of glioma stem cells and treatment of glioma by epigenetic inheritance at the level of post-translational protein modification by acetylation.Currently,most HDAC inhibitors as anti-tumor drugs work by targeting class I,Class II and Class IV HDAC.However,due to the inability to cross the blood-brain barrier,weak specificity and high toxicity,they are not effective in glioma.RGFP966 is an HDAC3 selective inhibitor that can be effectively distributed in the central nervous system with a cerebral: plasma concentration ratio of 0.45.It has been reported that the HDAC3 inhibitor RGFP966 has a protective effect on central nervous system degeneration and cerebral ischemia,indicating that RGFP966 can effectively cross the blood-brain barrier with high specificity and few side effects.This suggests that the HDAC3 selective inhibitor RGFP966 may effectively act on GSCs.In conclusion,selective inhibition of HDAC3 by RGFP966 can induce differentiation of glioma stem cells,inhibit proliferation,and reduce the degree of malignancy.Intervention therapy from the perspective of post-translational modification may provide a new direction for the treatment of glioma.Methods:(1)TCGA database was used to detect HDAC3 expression and immunohistochemistry was used to detect HDAC3 expression levels in human glioma samples.(2)In vitro experiments were performed to observe the inhibitory effect of HDAC3 gene expression(sh Hdac3)/activity(RGFP966)on glioma.Cell lines: mouse glioma stem cells CSC1589,CSC2078 and human glioma stem cells TS541.Experimental groups: negative control group,RGFP966 group or empty vector(EV)and sh Hdac3 group.Experimental conditions: proliferation conditions: Cells are cultured in NBM(Neurobasal media)culture medium,and growth factors are added into it without serum.Differentiation conditions: NBM(Neurobasal media)culture medium was added into 1% FBS without growth factor.Under proliferation conditions,CCK-8,growth counting,Brd U staining,and soft AGAR clone formation experiment were used to detect the effect on the proliferation ability of GSCs.The effect of neurosphere formation experiment on the self-renewal ability of GSCs was examined.Immunofluorescence staining,Western blot and q PCR were used to detect the effects of Nestin and GFAP,S-100?,Olig2 and Neu N on the differentiation of GSCs stem cells.(3)The effect of RGFP966 on the differentiation and proliferation of brain glioma stem cells was observed in vivo.Tumor stem cell TS541 was used to construct a mouse subcutaneous xenograft model.Balb/ C nu/nu mice were randomly divided into control group and RGFP966 group.The RGFP966 group received subcutaneous injection at about 1cm of the transplanted tumor three times a week.The control group was injected with the same amount of solvent by the above method.The mental ability,activity and eating state of the mice were observed every day.The tumor diameter and weight of the mice were observed and measured.Immunohistochemical staining was used to observe the effects of RGFP966 on proliferation and differentiation markers of subcutaneous transplantation of glioma stem cells.(4)To investigate the differentiation mechanism of GSCs induced by RGFP966/sh Hdac3 inhibition.In the differentiation state,the microarray was preliminarily screened for the effect of RGFP966 on the differentiation pathway of GSCs.The results showed that RGFP966 could inhibit multiple stem cell related pathways of GSCs,with TGF-? pathway as the most obvious one.The effect of RGFP966 on the self-renewal ability of TGF-?1 was detected by limited dilution method.Effect of RGFP966 on endogenous TGF-? expression level was detected by ELISA.Western blot and q PCR were used to detect the effect of RGFP966 on key molecules of TGF-? pathway.The influence of RGFP966 and sh Hdac3 on the expression level of acetylation and ubiquitination of Smad7 was detected by Western blot and IP/IB,and the acetylation site of Smad7 was detected by Ch IP.By overexpression and si RNA interference of Smad7,the effect of Smad7 on GSCs was detected by spheroidization experiment,Western blot,q PCR and other methods.Results:(1)HDAC3 was found to be a potential therapeutic target for glioma.TCGA results showed that the HDAC3 level in GBM was significantly higher than that in the normal control group.Immunohistochemistry showed that the expression level of HDAC3 was increased in human glioma,which was positively correlated with the malignant degree of glioma.(2)The effect of RGFP966/sh Hdac3 on the differentiation and proliferation of brain glioma stem cells was observed in vitro.CCK-8 results showed that RGFP966 inhibited the cell viability of GSCs in a dose-dependent manner.Growth counting results showed that inhibition of RGFP966/sh Hdac3 could inhibit the growth of GSCs.The results of Brd U staining showed that RGFP966 could down-regulate the expression level of Brd U.The results showed that RGFP966/sh Hdac3 could inhibit the clonal formation ability of GSCs.The results showed that RGFP966/sh Hdac3 could reduce the self-renewal ability of GSCs.Under differentiation conditions,immunofluorescence results showed that RGFP966 induced down-regulation of Nestin expression in GSCs and inhibited the stem cell characteristics of GSCs,leading to significant up-regulation of GFAP and S-100? expression levels,slight up-regulation of Neu N expression level,and significant down-regulation of Olig2 expression level,suggesting that inhibition of HDAC3 could induce GSCs differentiation into astrocytes.(3)The effect of RGFP966 on the differentiation and proliferation of brain glioma stem cells was observed in vivo.The tumor size and weight of mice were analyzed,and the results showed that the tumor volume of the RGFP966 group was significantly reduced compared with the control group.Immunohistochemical results showed that RGFP966 could reduce the expression of PCNA in tumor tissues,increase the expression of GFAP and S-100?,and down-regulate the expression of TGF-? RI and Sox2.At the same time,RGFP966 had no effect on body weight and no side effects such as organ damage.(4)To investigate the differentiation mechanism of GSCs induced by RGFP966/sh Hdac3 inhibition.In the differentiation state,the gene chip was preliminarily screened for the effect of RGFP966 on the differentiation pathway of GSCs.The results showed that RGFP966 could inhibit multiple stem cell related pathways of GSCs,with TGF-? pathway as the most obvious one.The results of limited dilution showed that RGFP966 inhibited the stimulation of TGF-? on the self-renewal ability of GSCs.ELISA results showed that RGFP966 had no significant effect on the expression level of endogenous TGF-? in GSCs.q PCR results showed that RGFP966 could induce down-regulation of GSCs TGF-? RI and Sox2 expression,and up-regulation of Smad7 expression.Western blot results showed that RGFP966 could inhibit the down-regulation of TGF-? RI,Sox2 and p-Smad2/3 in GSCs,suggesting that RGFP966 could inhibit the TGF-? pathway in GSCs.Smad7 is a negative regulator molecule of TGF-? pathway.Western blot results showed that under the action of RGFP966,the expression level of Smad7 continued to increase within 72 hours.IP/IB results showed that RGFP966/sh Hdac3 increased Smad7 acetylation level and decreased ubiquitin level.Ch IP results showed that RGFP966/sh Hdac3 increased the acetylation level of GSCs Smad7 in H3K27 domain.Overexpression of Smad7 inhibited the self-renewal ability of GSCs,and down-regulated the expressions of p-Smad2/3,TGF-? RI and Sox2.However,down-regulation of Smad7 significantly increased the self-renewal ability of GSCs and up-regulated p-Smad2/3,TGF-? RI and Sox2 expression,and the application of RGFP966 could not be reversed these results.The results suggested that RGFP966/sh Hdac3 could inhibit TGF-? pathway through Smad7.Conclusion:Inhibition of HDAC3 expression/activity can inhibit the proliferation,self-renewal ability and stem cell characteristics of GSCs,and induce GSCs to differentiate into astrocytes.Inhabition of HDAC3 expression/activity increased the acetylation level of SMAD7,blocking the ubiquitination of SMAD7,and inhibited the TGF-? pathway by activating SMAD7,thereby inhibiting the characteristics of stem cell ability.RGFP966 can be used to intervene from the perspective of post-translational modification,providing a new direction for the treatment of glioma.HDAC3 is a potential therapeutic target for glioma. |
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