2017-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/691971摘要：惡性神經膠質瘤是最常見的原發性腦瘤，即使以手術、放射線治療、及化學治療等多方面的治療方法，惡性神經膠質瘤病人的預後還是不好。不正常的染色質(chromatin)的調節及表觀遺傳(epigenetic)的修飾與癌症的發生有關，Zinc finger MYND-type containing 8 (ZMYND8)是轉錄因子，會與histone H3交互作用，也是染色質的reader及effector，它與子宮頸癌、大腸直腸癌、表皮T細胞淋巴癌及急性紅白血病(acute erythroid leukemia)有關聯，ZMYND8的表現在斑馬魚的攝護腺癌異體移植及人的攝護腺癌標本有增加的現象，它也與腫瘤細胞的侵襲及腫瘤的血管生成有關，但動物實驗發現它在乳癌及攝護腺癌有抑制腫瘤的作用，所以它在癌症的角色仍不清楚。小孩及年輕人的多型神經膠母細胞瘤(glioblastoma multiforme, GBM)常有與histone H3.3的結構有關的α-thalassemia/mental retardationsyndrome X-linked (ATRX)、death-domain associated protein (DAXX)、及histone H3.3變異型H3F3A的基因突變；此外，神經膠質瘤的polycomb repressive complex 2、epidermal growth factor receptor及isocitratedehydrogenase 1等都有異常的基因表現及表觀遺傳的變化，而此三者都與神經膠質瘤的生成有關。這些現象顯示不正常的表觀遺傳調節及histone的修飾，尤其是H3，可能是神經膠質瘤的腫瘤生成的重要因子，因此ZMYND8可能在神經膠質瘤的腫瘤生成扮演重要角色。在此三年計畫中，我們將探討ZMYND8在神經膠質瘤的腫瘤生成的角色。首先，我們研究ZMYND8在神經膠質瘤細胞株(人的T98G、A172、U87MG及U138MG，及大鼠RT-2細胞株)的表現，我們的初步研究(real time PCR及western blot analysis)發現這些神經膠質瘤細胞株的ZMYND8的表現都比計畫主持人的白血球的表現低。然後將繼續探討ZMYND8的過度表現或以siRNA抑制ZMYND8的表現對於神經膠質瘤細胞的增殖分裂、集落形成能力(colony forming ability)、凋亡、侵襲性等腫瘤細胞行為的影響。從這些資料我們將可了解ZMYND8的作用是抑制或促進腫瘤生成，接著我們會探討ZMYND8在神經膠質瘤細胞的表觀遺傳作用，並找出被ZMYND8調節的基因，再從當中挑出與腫瘤發生相關的主要基因，研究他們是否與ZMYND8的功能有關，再以ZMYND8基因的區域截斷式突變型(domain-truncatedmutants)探討其分子機轉，確認ZMYND8基因的作用區域，並確認此區域的作用。最後我們將進行動物實驗，探討ZMYND8過度表現(或以siRNA抑制ZMYND8)對於神經膠質瘤細胞在大鼠皮下及腦部的腫瘤生成的影響。文獻上並無類似的研究報告，本計畫的結果將幫助我們了解ZMYND8在神經膠質瘤的腫瘤生成的角色，將會為神經膠質瘤的治療提供一新的途徑。<br> Abstract: Malignant glioma is the most common primary brain tumor and patients with malignant gliomas havepoor prognoses. Aberrant chromatin regulation and epigenetic histone modification are related to cancerdevelopment. Zinc finger MYND-type containing 8 (ZMYND8), a transcriptional factor, a histoneH3-interacting protein and also a putative chromatin reader/effector, has been found to be associated withcervical and colorectal cancers, acute erythroid leukemia, and cutaneous T-cell lymphoma. ZMYND8expression is upregulated in both zebrafish prostate cancer xenografts and prostate cancer specimens frompatients, and can affect tumor cell migration and invasion, and tumor angiogenesis. In contrast, ZMYND8exerts tumor suppression effect on breast and prostate cancers in vivo. Therefore, the actual role of ZMYND8in cancers including glioma is unclear. Aberrant DNA hypomethylation events and H3 histone modificationare considered to activate normally silenced promoters within gene bodies, and might thus lead to expressionof various oncogenic proteins in glioblastoma multiforme (GBM). In addition, α-thalassemia/mentalretardation syndrome X-linked (ATRX) and death-domain associated protein (DAXX) mutations arecommonly seen in pediatric gliomas, and they encode two subunits of a chromatin remodeling complexrequired for H3.3 incorporation at pericentric heterochromatin and telomeres. Pediatric GBMs and pontinegliomas often show mutations in H3F3A gene (H3.3 encoding gene). Furthermore, aberrant regulation ofgene expression and epigenetic mutations are noted in polycomb repressive complex 2, epidermal growthfactor receptor and isocitrate dehydrogenase 1, which are highly associated with gliomas. All these findingsindicate that aberrant epigenetic regulation and histone modifications especially H3 may be important for theoccurrence of gliomas. Therefore, ZMYND8 might play a role in the tumorigenesis of gliomas.In this project, we intend to study the role of ZMYND8 in the tumorigenesis of gliomas. Firstly, theZMYND8 expression in the glioma cell lines is studied. In our preliminary study, the expression ofZMYND8 in various glioma cell lines was lower than that in the principal investigator’s white blood cells(WBC) using real time polymerase chain reaction and western blot analysis. Then the effects of ZMYND8 onthe glioma cells are studied by ZMYND8 blockade or overexpression, and the tumor cell proliferation,colony forming ability, apoptosis, and invasiveness are observed. From these data, the functions of ZMYND8(tumor suppression or tumor promotion) will be determined. Then the epigenetic function of ZMYND8 andthe ZMYND8-regulated genes will be investigated. The key ZMYND8-regulayed genes will be identifiedand subjected to further investigation about their roles in the ZMYND8-meidated functions. The molecularmechanism of ZMYND8 is explored using domain-truncated mutants of ZMYND8 to identify the interactingdomains of ZMYND8. Finally, animal experiments will be done. The rat RT-2 glioma cells with ZMYND8overexpression or siRNA blockade will be implanted to the subcutis or brain of the rats, and the animalsurvival and tumor growth will be compared. There is no similar report in the literature. The results in thisproject will help us to understand the role of ZMYND8 in the tumorigenesis of gliomas. It might open a newway to the treatment of gliomas.ZMYND8神經膠質瘤腫瘤生成ZMYND8gliomatumorigenesis