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,因此,充分了解神經膠質瘤的生成、生長及侵襲的病態 生理及作用機轉,進而發展出有效的治療方法是必要的。BS69是一多區域(multidomain)蛋白質,它的 基因位於第十對染色體,BS69的作用與染色質的重塑(chromatin remodeling)有關,是正常細胞生長程 序的感應器4S69基因位於人類癌症常發生缺失(deletion)的區域,且在幾種癌症及白血病其表現有降 低的現象;BS69可與一些轉錄因子例如c-Myb、Ets2、Myc-related MGA protein作用,扮演轉錄抑制 者的角色,而這些轉錄因子都與腫瘤的發生有密切關係,且BS69的過度表現可抑制小鼠乳癌的腫瘤 生長,這些資料顯示BS69可能是一腫瘤抑制者,不過其真正的角色仍不清楚。文獻上並無研究報告 探討BS69在神經膠質瘤的腫瘤發生的角色,但是有些資料顯示BS69可能與神經膠質瘤的發生有關, BS69可辨識histone H3.3上的H3K36 trimethyl mark (H3.3K36me3),此外,小孩及年輕人的神經膠母 細胞瘤(glioblastoma multiforme, GBM)常有 histone H3.3 變異型 H3F3A 的基因突變,因 H3F3A G34 突 變會造成MYCN表現的增加,而MYCN若表現在發育中的大腦皮質會引發神經膠母細胞瘤,所以BS69 可能在神經膠質瘤的腫瘤發生扮演重要的角色。在此三年計晝中,我們將探討BS6在神經膠質瘤的腫瘤生成的角色。首先,我們將研究BS69在四 種神經膠質瘤細胞株(人的T98G、A172及U87MG,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及U2OS骨肉瘤(osteosarcoma)細胞(positive control)的BS69的表現都比計晝主持人的白血球的表現低。接著我們將探討BS69過度表現或以siRNA 抑制BS69對於神經膠質瘤細胞的生物行為包括形狀、聚集(aggregation)、黏著(adhesion)、增殖分裂、 调亡、形成球體的活力(sphere-forming activity)、侵襲力等的影響。從這些資料我們將可了解BS69的功 能是抑制或促進腫瘤增生,這將作為後續研究的基礎。然後我們會以array screening來研究與BS69相關 的调亡因子及細胞週期調節因子,並以western blot及real time PCR來確認其結果。同時我們也將探討 BS69相關的訊息通路、腫瘤抑制蛋白質(tumor suppressor protein)、腫瘤促進蛋白質(oncoprotein)、 histones的表現、細胞凋亡通路及其上下游的通路。此外我們也將研究在神經膠質瘤細胞中與BS69的 作用有關的microRNAs。最後我們將進行動物實驗,探討BS69過度表現(或以siRNA抑制BS69)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,也將研究BS69及相關的訊息通路在腦瘤組織的表 現。文獻上並無類似的研究報告,本計晝的結果將幫助我們了解BS69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。
Abstract: Malignant glioma is the most common primary brain tumor. Patients with malignant gliomas have poor prognoses, even using multidisciplinary treatment strategies including surgery, radiotherapy, and chemotherapy, Therefore, understanding the pathophysiology and mechanisms of tumor formation, growth and invasion; and developing a better therapeutic strategy for malignant brain tumors are mandatory.BS69 is a multidomain protein with the gene located at chromosome 10. BS69 plays a role in chromatin remodeling and functions as a sensor in the normal cellular growth programs. The location of BS69 gene is in a region frequently deleted in human cancers and the BS69 expression is frequently downregulated or deleted in several human cancers and leukemia. In addition, BS69 acts as a transcriptional repressor in association with a variety of transcriptional factors, such as c-Myb, Ets2, Myc-related MGA proteins, and these factors are closely associated with tumorigenic process. Overexpression of BS69 can suppress tumor growth of the breast cancers in mice. These data suggest BS69 might act as a tumor suppressor, however, the actual role of BS69 in tumorigenesis is still unclear. In the literature, there is no report investigating the role of BS69 in the tumorigenesis of gliomas. However, some data suggest BS69 might play a role in the gliomagenesis. BS69 specifically recognizes histone H3K36 trimethyl mark (H3K36me3) on H3.3 (H3.3K36me3). Children and young adults with glioblastoma multiforme (GBM) often have mutations in the gene encoding the histone H3 .3 variant H3F3A, and the H3F3A G34 mutation causes profound upregulation of MYCN, a potent oncogene that can cause GBMs when expressed in the developmental cortex. Therefore, BS69 may play an important role in the tumorigenesis of gliomas.In this 3-year project, we intend to investigate the role of BS69 in the tumorigenesis of gliomas. Firstly, the expression of BS69 in three human (T98G,A172 and U87MG) and rat RT-2 glioma cell lines are studied. In our preliminary study, we had tested the relative expression level of BS69 in the available glioma cell lines, U2OS cells (positive control) and principal investigator5s (PI5s) white blood cells (WBC) using real time polymerase chain reaction and western blot analysis, and the results revealed all the tumor cells had relatively lower BS69 expression level thanPI’s WBC. Subsequently, the effects of BS69 overexpression or knockdown by siRNA on the morphology, proliferation and apoptosis, aggregation, adhesion, stem cell-like sphere formation and invasiveness of the glioma cells will be investigated. From the in vitro study, the function of BS69 (tumor suppression or tumor promotion) will be defined. Then the possible BS69-related apoptotic factors and cell cycle regulators in the glioma cells will be studied by array screening; and the results will be confirmed by western blot and real time polymerase chain reaction. Next, we will explore the expression of possible BS69-related tumor suppressor proteins, oncoproteins, histones, apoptosis pathways, and other signaling pathways. In addition, the microRNAs involved in the action of BS69 in glioma cells will be studied. Finally we will investigate the effects of overexpression (or knockdown) of BS69 on the in v^^vo subcutaneous and intracerebral tumorigenesis in rats, including tumor growth rate, animal survival time, the expression of BS69 and microRNAs, and related signaling pathways in the tumor specimens. There is no similar report in the literature. The results in this project will help us to understand the role of BS69 in the tumorigenesis of gliomas. It might open a new way to the treatment of gliomas.