摘要:原發性腦瘤約有一半源自神經膠細胞,稱為神經膠瘤。依據世界衛生組織的分類,神經膠瘤可分為高惡度神經膠瘤與低惡度神經膠瘤,其中,高惡度神經膠瘤佔所有顱內腫瘤的40%以上。近年來針對腦瘤採取多重處理與多重治療模式,部分改善了腦瘤的預後,然而,惡性神經膠瘤病患的存活期仍相當有限。神經膠瘤的病理惡性度依舊是預測惡性神經膠瘤病患預後的主要因子。最近有研究顯示病患對於放射治療與烷基化療藥物具有抗性可能是導致神經膠瘤病患預後不佳的因素之一。因為惡性神經膠瘤在分子層次上存在相當的異質性,所以,某些治療方式或許只對特定的少數病患的病情有幫助,但對其餘的病患卻是沒有療效。為增進神經膠瘤病患的預後,研發新的病患預後評估機制將有助於現行標準的治療模式。最近的研究報告發現,微型核醣核酸(microRNA)的表現剖繪(expression profile)可能較基因表現剖繪更能區分癌症的亞型。微型核醣核酸是一種長度約18-25核苷酸的內生性核醣核酸,它可以透過轉錄後機制抑制數以百計下游基因的蛋白質表現,藉此方式,微型核醣核酸調控許多基礎的細胞反應,包括,細胞生長、分化、外在壓力反應、細胞死亡等。最近研究顯示,微型核醣核酸的異常表現與許多種癌症的某些特徵有關連性,如癌症生成、分化程度與疾病預後,顯示微型核醣核酸可以扮演致癌基因或是癌症抑制基因,其表現在癌症的發生及進程上扮演重要的角色。西方國家在個別微型核醣核酸的表現與神經膠瘤預後的研究報告不多,而且臨床上顯示東方人的基因表現以及對藥物的感受性在某些情況下可能不同,所以以微型核醣核酸為基礎的預後診斷基因標記及針對台灣族群所發展的一套可靠的微型核醣核酸生物標記值得探討與建立。本計畫我們擬利用微型核醣核酸低密度微陣列(microRNA Low density Array)分析40個冷凍神經膠瘤檢體中365個微型核醣核酸的表現量並結合其臨床數據,透過Cox回歸法找出與疾病預後有顯著相關的微型核醣核酸群。此外,針對12株對腦瘤化療藥物carmustine (BCNU)與temozolomide (TMZ)具有不同抗藥性的神經膠瘤細胞株,進行微型核醣核酸表現剖繪與統計分析,以期發現與抗藥性相關的微型核醣核酸。神經膠瘤石蠟包埋檢體將用以驗證這兩種印記(預後微型核醣核酸印記與抗藥性微型核醣核酸印記)對於病患預後與抗藥性的預測準確性。我們的初步研究結果已證實以微型核醣核酸低密度微陣列偵測神經膠瘤檢體中微型核醣核酸的作法是可行的,同時,7株神經膠瘤細胞株已完成對BCNU與TMZ的抗藥性測定。在未來的計畫中,將運用生物資訊工具鑑定這些與神經膠瘤預後和抗藥性相關之微型核醣核酸的標的基因為何,並將這些微型核醣核酸的標的基因選殖至報導基因載體並送入細胞表現,利用西方墨點法及報導基因分析證明其間的調控關係。此外,亦將微型核醣核酸選殖至表現載體並利用活體外及活體內的研究方法來定性這些微型核醣核酸的功能,例如對細胞侵襲、移動、群聚形成、血管新生、腫瘤生成和抗藥性等能力的影響。經由這些努力,我們預期可獲得會影響神經膠瘤進程及抗藥性的重要微型核醣核酸及其標的基因,這些微型核醣核酸除了可被利用來當作預後診斷的標記分子外,未來也可做為治療的標的。
Abstract: Approximately half of primary brain tumors arise from glial cells, which are named as gliomas. Gliomas are categorizes by the World Health Organization (WHO) as either low-grade or high-grade. High-grade glioma (HGG) comprises more than 40% of all intracranial tumors. Despite recent successes in the treatment of cancer with multidisciplinary multimodal treatment approaches, the duration of survival for patients with malignant glioma remains limited. The most important determinant of prognosis in malignant glioma is largely its grading. Recently, undesirable prognosis for patients with malignant glioma has been attributed to resistance to radiotherapy and alkylating chemotherapeutic agents. Malignant glioma is a molecularly heterogeneous group, therefore it is possible that some treatments may be highly beneficial to a select few patients, yet ineffective in many others. To improve the clinical outcome of patients having a malignant glioma, new strategies need to be developed and evaluated to complement the current standard therapies. Recently, the expression profiling of microRNAs may be a more accurate method of classifying cancer subtype than using the expression profiles of protein coding genes. MicroRNAs are endogenous small non-protein-coding RNAs (18-25 nucleotides) that conduct the post-transcriptional repression of hundreds of their target proteins. They regulate many kinds of basic cellular processes including proliferation, differentiation, stress response, and cell death. Recent evidence has shown that deregulation of microRNAs correlates with certain features of diverse cancers (such as metastasis, tumorigenesis, differentiation status, and outcome of tumor patients) and indicates that microRNAs can act as oncogenes or tumor suppressors and alterations in microRNA expression may play a critical role in the cancer initiation and progression. Although the correlation between individual microRNA expression and the outcome of brain cancers has been studied in western population in a few reports, neither microRNA signature with multiple microRNAs nor a panel of reliable markers for its prognosis in Taiwan has been established.In this project, we plan to profile 365 microRNAs from 40 glioma tissues by microRNA Low density Array. Associated with the clinical information, we will use the Cox regression to identify a unique signature containing several microRNAs whose expression is significantly correlated with the outcome of patients with glioma. On the other hand, the microRNAs associated with drug resistance of glioma will be identified using microRNA profiling, Cox regression and a panel of 12 brain cancer cell lines with different drug susceptibility of carmustine (BCNU) and temozolomide (TMZ). Both prognostic and drug resistance microRNA signatures identified will be applied to formalin fixed paraffin embedded (FFPE) glioma tissues to verify the predictors of prognosis and chemotherapeutic regiments. In the preliminary study, we have demonstrated it is feasible to detect the microRNA profiles in frozen glioma specimens. The drug susceptibility is also characterized in seven glioma cell lines.Subsequently, the relationship between microRNA candidates and their target genes will be identified by bioinformatics and further validated by Western blot and luciferase reporter assays. In addition, the microRNAs will be constructed into an expression vector and subjected to the functional characterization by in vitro and in vivo approaches such as invasion, migration, anchorage independent/dependent growth, angiogenesis and tumorigenesis as well as drug resistance. Through our efforts, we anticipate identifying critical microRNAs and their target genes responsible for glioma progression and drug resistance, which might be employed as prognostic markers or serve as therapeutic targets in the future purpose.