【作者】 梁航；

【导师】 张灵；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2020， 博士

【摘要】 R背景:恶性胶质瘤是最常见的、致死率最高的颅内恶性肿瘤,侵袭性强,耐药性强,复发率高。胶质瘤干细胞(Glioma stem cells,GSCs)是胶质瘤细胞的一个亚类,根据肿瘤干细胞假说,GSCs是一类具有干细胞样特征,具有很强的自我更新和增殖能力的肿瘤细胞,是诱导胶质瘤发生、造成胶质瘤放化疗抵抗以及肿瘤复发的重要原因之一。传统的放化疗旨在杀死快速增殖的肿瘤细胞。而肿瘤干细胞是相对静止的、并可将药物转运至细胞外,降低药物作用。同时肿瘤干细胞可通过维持低水平活性氧来减少放疗引起的损伤。因此,传统的放化疗对肿瘤干细胞效果欠佳造成肿瘤复发。因此,靶向GSCs进行治疗可能是治疗恶性胶质瘤的新策略。目前对于肿瘤干细胞主要的策略是改变其自我更新相关信号通路以及改变其微环境通路,但针对于胶质瘤干细胞的研究仍较少。异常表观遗传学修饰在肿瘤表型的维持中发挥着重要作用。对于GBM的发病机制来讲,胶质瘤干细胞可能存在异常的表观遗传修饰而使其维持干细胞状态不分化或分化不完全。但与基因突变不同的是,表观遗传修饰异常过程常常是可逆的。如果通过表观遗传学方法诱导胶质瘤干细胞向成熟胶质细胞分化,有望降低肿瘤恶性程度,并抑制肿瘤干细胞放化疗抵抗。组蛋白乙酰化转移酶(Histone acetyltransferase,HATs)和组蛋白去乙酰化酶(histone deacetylase,HDACs)在恶性胶质瘤中常存在着失衡。组蛋白去乙酰化酶3(histone deacetylase3,HDAC3)的过表达与胶质瘤密切相关,HDAC3过表达可引起组蛋白去乙酰化水平增强,导致一些抑癌基因如p53、p21等表达降低,从而导致肿瘤的发生。组蛋白去乙酰化酶抑制剂可抑制组蛋白去乙酰化酶的作用,从而起到治疗肿瘤的目的。同时,通过表观遗传学治疗,在蛋白翻译后修饰水平进行干预,作用更为精准。有研究表明,抑制HDAC3可促进肿瘤细胞凋亡和细胞周期阻滞,可通过促进恶性血液肿瘤分化抑制肿瘤增殖,但对于胶质瘤干细胞作用并不明确。在前期工作中,本研究组在对胶质瘤干细胞的表观遗传学染色质调控子的筛查中发现,下调HDAC3基因的表达,除可抑制GSCs增殖外,可明显促进其分化。因此推测通过抑制HDAC3可诱导胶质瘤干细胞分化、并且通过促进蛋白翻译后乙酰化修饰治疗脑胶质瘤。目前,大多数作为抗肿瘤药物的HDAC抑制剂是通过靶向I类、II类和IV类HDAC来发挥作用,但是存在无法透过血脑屏障、特异性不强、毒性大等问题,对胶质瘤效果欠佳。RGFP966是HDAC3选择性抑制剂,可在中枢神经系统有效分布,脑:血浆浓度比值为0.45。有文献表明HDAC3抑制剂RGFP966对于中枢神经系统退行性病变、脑缺血有保护作用,这说明RGFP966可有效透过血脑屏障,特异性高,副作用小。综上,利用RGFP966选择性抑制HDAC3诱导胶质瘤干细胞分化、抑制增殖,降低其恶性程度,从翻译后修饰角度干预治疗,可能为胶质瘤治疗提供新的治疗靶点。目的:通过应用HDAC3选择性抑制剂RGFP966以及sh Hdac3抑制HDAC3活性或表达,通过体内外实验观察抑制HDAC3对胶质瘤干细胞增殖能力、干细胞特性、分化能力的影响,探讨其可能的机制。方法:(1)利用TCGA数据库检测HDAC3表达情况以及免疫组化检测HDAC3在人脑胶质瘤样本中表达水平。(2)体外实验观察抑制HDAC3活性(RGFP966)/表达(sh Hdac3)对脑胶质瘤干细胞增殖及分化的影响。实验细胞系:鼠源胶质瘤干细胞CSC1589,CSC2078以及人源胶质瘤干细胞TS541。实验分组:DMSO组、RGFP966组或empty vector(EV)组、sh Hdac3组。实验条件:增殖条件(维持干细胞特性):细胞培养于NBM(Neurobasal media)培养基,加入生长因子,无血清;分化条件(促进干细胞分化):NBM(Neurobasal media)培养基,加入1%FBS,不含生长因子。在增殖条件下,利用CCK-8、生长计数、Brd U染色、软琼脂克隆形成实验检测对GSCs增殖能力的影响;神经球成球实验检测对GSCs自我更新能力的影响;在分化条件下,利用免疫荧光染色、Western blot、q PCR检测GSCs干细胞标志物Nestin及分化标志物GFAP,S-100β,Olig2、Neu N等的表达。(3)体内实验观察RGFP966对脑胶质瘤干细胞增殖及分化的影响。利用人源胶质瘤干细胞TS541,构建小鼠皮下移植瘤模型,将Balb/C nu/nu小鼠随机分为对照组及RGFP966组。RGFP966组在移植瘤约1cm处进行皮下注射给药,一周三次。对照组采用上述方法注射等量药物溶剂。每天观察小鼠的精神状态、活动、进食情况,观察并测量肿瘤直径及称量小鼠体重。免疫组织化学染色观察RGFP966对胶质瘤干细胞皮下移植瘤增殖、分化标志物的影响。(4)探讨抑制RGFP966/sh Hdac3诱导GSCs分化的机制。基因芯片初步筛查RGFP966对GSCs分化相关通路的影响;有限稀释法检测RGFP966对TGF-β1作用下GSCs自我更新能力影响;ELISA检测RGFP966对内源性TGF-β表达水平的影响。Western blot、q PCR检测RGFP966对TGF-β通路关键分子的影响。通过Western blot、IP/IB检测RGFP966、sh Hdac3对Smad7表达水平、乙酰化、泛素化水平影响,利用Ch IP检测Smad7发生乙酰化位点。通过过表达Smad7、si RNA干扰技术下调Smad7,利用神经球成球能力实验、Western blot、q PCR等方法检测Smad7对GSCs的影响。结果:(1)发现HDAC3可能是脑胶质瘤潜在的治疗靶点。TCGA结果表明GBM中HDAC3水平较正常对照组明显升高;免疫组化表明人脑胶质瘤中HDAC3表达水平升高,并与胶质瘤恶性程度呈正相关。(2)体外实验观察RGFP966/sh Hdac3对脑胶质瘤干细胞增殖及分化的影响。在增殖条件下,CCK-8结果表明,RGFP966可抑制GSCs的细胞活力,存在剂量依赖性;生长计数结果表明RGFP966/sh Hdac3可抑制GSCs的生长能力;Brd U染色结果表明RGFP966可下调GSCs Brd U表达水平;软琼脂克隆形成实验结果表明RGFP966/sh Hdac3可抑制GSCs克隆形成能力;神经球成球能力实验结果表明RGFP966/sh Hdac3可降低GSCs自我更新能力。在分化条件下,免疫荧光结果表明RGFP966引起GSCs中Nestin表达下调,抑制GSCs的干细胞特性,可导致GFAP、S-100β表达水平明显上调,Neu N表达水平轻度上调,Olig2表达水平明显下调,提示抑制HDAC3可诱导GSCs向星形胶质细胞分化。(3)体内实验观察RGFP966对脑胶质瘤干细胞增殖及分化的影响。对肿瘤大小和小鼠体重进行测量及分析,结果表明,与对照组相比,RGFP966组肿瘤体积明显缩小。免疫组化结果表明,RGFP966可降低肿瘤组织中PCNA表达,增加GFAP以及S-100β表达,使TGF-βRI、Sox2表达下调。应用RGFP966对小鼠体重无影响,无脏器损伤等副作用。(4)探讨RGFP966/sh Hdac3诱导GSCs分化机制。在分化状态下,基因芯片初步筛查RGFP966对GSCs分化通路影响,结果表明,RGFP966可抑制GSCs多条干细胞相关通路,以TGF-β通路抑制最明显。有限稀释法结果表明RGFP966可抑制TGF-β1对GSCs自我更新能力的激活作用。ELISA结果表明RGFP966对GSCs内源性TGF-β表达水平无明显影响;q PCR结果表明,RGFP966可引起GSCs TGF-βRI及Sox2表达下调,Smad7表达上调;Western blot结果表明,RGFP966可引起GSCs中TGF-βRI、Sox2及p-Smad2/3表达下调,提示RGFP966可抑制TGF-β通路。Smad7是TGF-β通路负调控分子,Western blot结果表明,在RGFP966作用下,Smad7表达水平在72h内持续增高。免疫沉淀结果表明,RGFP966/sh Hdac3使Smad7乙酰化水平增加,泛素化水平降低。染色质免疫共沉淀实验(Ch IP)结果表明,RGFP966/sh Hdac3使Smad7与H3K27乙酰化蛋白结合。过表达Smad7可抑制GSCs的自我更新能力,使p-Smad2/3、TGF-βRI及Sox2表达下调;而下调Smad7会增加GSCs的自我更新能力,使p-Smad2/3、TGF-βRI及Sox2表达上调,应用RGFP966不能逆转,以上结果提示RGFP966/sh Hdac3可通过Smad7抑制TGF-β通路。结论:(1)下调HDAC3活性/表达可抑制GSCs增殖、自我更新能力以及干细胞特性,并诱导GSCs向星形胶质细胞分化,降低肿瘤恶性程度及致瘤性。(2)抑制HDAC3活性/表达可增加GSCs SMAD7乙酰化水平,避免SMAD7被泛素化降解,从而通过SMAD7/TGF-β通路抑制GSCs干细胞特性,并诱导GSCs分化。(3)利用HDAC3选择性抑制剂RGFP966可从翻译后修饰角度干预治疗,为胶质瘤治疗提供新的治疗靶点。更多还原

【Abstract】 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-100β,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-β pathway as the most obvious one.The effect of RGFP966 on the self-renewal ability of TGF-β1 was detected by limited dilution method.Effect of RGFP966 on endogenous TGF-β expression level was detected by ELISA.Western blot and q PCR were used to detect the effect of RGFP966 on key molecules of TGF-β 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-100β 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-100β,and down-regulate the expression of TGF-β 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-β pathway as the most obvious one.The results of limited dilution showed that RGFP966 inhibited the stimulation of TGF-β on the self-renewal ability of GSCs.ELISA results showed that RGFP966 had no significant effect on the expression level of endogenous TGF-β in GSCs.q PCR results showed that RGFP966 could induce down-regulation of GSCs TGF-β RI and Sox2 expression,and up-regulation of Smad7 expression.Western blot results showed that RGFP966 could inhibit the down-regulation of TGF-β RI,Sox2 and p-Smad2/3 in GSCs,suggesting that RGFP966 could inhibit the TGF-β pathway in GSCs.Smad7 is a negative regulator molecule of TGF-β 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-β RI and Sox2.However,down-regulation of Smad7 significantly increased the self-renewal ability of GSCs and up-regulated p-Smad2/3,TGF-β 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-β 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-β 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.更多还原