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標題: Identification and functional characterization of microRNAs that involved in lung cancer progression and metastasis
作者: 陳健尉
關鍵字: NSCLC;基礎研究;基礎醫學類, 生物技術, 醫學技術;metastasis;expression profiling;microRNA;microarray;two-dimensional difference gel electrophoresis;非小細胞肺癌;癌轉移;基因表現剖繪;微核醣核酸;微陣列;二次元差異膠體電泳
Lung cancer, predominantly non-small-cell lung cancer (NSCLC), is the most common cause of cancer deaths worldwide, and metastasis is the major cause leading to mortality for cancer patients. The relapse rate among patients with early-stage NSCLC is 40% within 5 years after potentially curative treatment. The current staging system for NSCLC is inadequate for predicting the outcome of treatment. mRNA expression profiling can be useful in the classification or predicting the prognosis of various types of cancer, including lung cancer. However, the expression profiling of microRNAs may be a more accurate method of classifying cancer subtype than using the expression profiles of protein coding genes was recently reported. MicroRNAs are a new class of small non-protein-coding RNAs that can act as endogenous RNA interference. MicroRNAs can post-transcriptionally regulate the expression of their target genes and control a wide range of biological functions such as cellular proliferation, differentiation and apoptosis. Recent evidences indicate that microRNAs may function as tumor suppressors or oncogenes 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 lung cancers has been studied in western population, 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 identify those microRNAs by microarray and real-time RT-PCR approaches. In the preliminary study, we have demonstrated it is feasible to develop a signature for the prediction of treatment outcome of NSCLC. The number of microRNA and case number will be increased to make the results more comprehensive and more robust. Subsequently, the target proteins of these microRNA candidates will be identified by two-dimensional difference gel electrophoresis/mass spectrometry and bioinformatics, either by homemade ( or publicly available tools, and further validated by Western blotting and luciferase reporter assays. In addition, the microRNAs will be constructed into expression or silencing vector and subjected to the functional characterization by in vitro and in vivo approaches such as invasion, migration, anchorage independent/dependent growth, tumorigenesis, and generation of knockout mice for the purpose of in vivo physiological function of microRNAs. Finally, the critical microRNAs and their targets will be applied to formalin fixed paraffin embedded tissues, and then generate a prognostic predictor. In addition, the animal model of lentivirus-based gene therapy will be established and employed to evaluate the curative potential of these identified microRNAs on lung cancer. Through our efforts, we anticipate identifying critical microRNAs and their target genes responsible for lung cancers progression and metastasis, which might be employed as prognostic markers, as well as serve as therapeutic targets in the future purpose.

肺癌,主要是非小細胞肺癌,為當今世上最常見的致死癌症之ㄧ,而癌轉移則是導致癌症病患死亡的主因。對早期的非小細胞肺癌病人而言,經治療後的五年疾病復發率約為40%,而目前的腫瘤分期系統並不足以預測其治療結果。基因表現剖繪(mRNA expression profiling)有助於許多不同癌症的分類或預測,其中也包括肺癌。然而,最近的研究報告顯示,微核醣核酸(microRNA)的表現剖繪可能較基因表現剖繪更能區分癌症的亞型。microRNA是新發現的ㄧ種不會轉譯成蛋白質的小分子核醣核酸,在細胞內具有降低訊息核醣核酸(mRNA)轉譯為蛋白質的能力。microRNA可調控數以百計的標的mRNA,並控制許多生物功能,例如細胞增殖、分化及凋亡。最近的證據指出,microRNA可能具有腫瘤抑制基因或致癌基因的功能,其表現在癌症的發生及進程上扮演重要的角色。雖然西方國家在個別microRNA的表現與肺癌的預後上已有著墨,但是以microRNA為基礎的預後診斷基因印記(gene signature)及針對台灣族群所發展的一套可靠的microRNA生物標記都有待建立。在此計畫中,我們將利用基因微陣列及即時定量反轉錄聚合酶連鎖反應等方法找出這些microRNAs。初步的研究結果已證實發展microRNA基因印記以預測肺癌治療結果的作法是可行的。在未來的計畫中,將增加microRNA的數目及實驗樣本數並進行統計測試以確保結果的廣泛性與正確性。接下來將這些microRNAs選殖至表現載體並送入細胞表現,利用二次元差異膠體電泳(two-dimensional difference gel electrophoresis)/質譜儀、西方墨點法、報導基因分析及自行開發(或網路上公開之生物資訊工具,以鑑定這些與預後相關之microRNAs的標的蛋白或基因為何。此外,亦將利用試管內及活體內的研究方法來定性這些microRNAs的功能,例如對細胞侵襲、移動、群聚(colony formation)、腫瘤形成等能力的影響,並建立基因剔除小鼠以觀察其對完整生物個體之生理及病理的影響。最後,將嘗試利用這些microRNAs及其標的基因來分析石蠟包埋的檢體,並建立預後預測模式;此外亦將建立以慢病毒為基礎的基因治療(lentivirus-based gene therapy)動物模式,以評估所鑑定的microRNAs是否具有疾病治療的潛力。經由這些努力,我們預期可獲得會影響肺癌進程及轉移的重要microRNAs及其標的蛋白或基因,這些microRNAs除了可被利用來當作預後診斷的標記分子外,在未來也可做為治療的標的。
其他識別: DOH97-TD-G-111-017
Appears in Collections:生命科學系所

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