利用FISH的方法來偵測Large Non-coding RNA在肝癌的表現

Detected the Expression of Large Non-coding RNA on Hepatocellular Carcinoma (HCC) using the FISH Method

林莉莉(Li-Li Lin)

指導教授 ： 張建國

高雄醫學大學/醫學院/醫學研究所/碩士(2010年)

摘要

在人類的基因體中，約有小於2%的鹼基可表現出蛋白質，其他大於90%以上的區域可轉錄但不能轉譯成蛋白質的RNA，稱為非編碼RNA(non-coding RNA ；ncRNA)。 其中以rRNA、tRNA、miRNA及snRNA等有較多的研究外，ncRNA的研究相當少。而與癌症有關的研究除了miRNA及少數的ncRNA外，其他的也並不多，特別是非常長的ncRNA (>5kb) 在癌症形成過程所佔角色的相關研究更少。本研究的目的是利用原位雜交技術 (FISH) 分析比較ncRNA在肝腫瘤細胞及其鄰近正常細胞的表現。
在本篇實驗中，利用生物資訊的方法得到40個大於5000bp以上的large ncRNA，利用即時定量聚合酶連鎖反應（Real-time quantitative Polymerase chain reaction；Real-time quantitative PCR）分析這些large ncRNA在肝癌的表現，結果得到4個large ncRNA，其HCC的癌細胞與其鄰近正常細胞確有差異表現。
我們進一步利用原位雜交技術(fluorescence in situ hybridization；FISH)染色的石蠟切片，在螢光顯微鏡下，HCC的癌細胞與其鄰近正常細胞所呈現的情形，和即時定量聚合酶連鎖反應(Real-time quantitative PCR )所呈現結果是一致的，並可得知ncRNA在cell中的位置。由此可見，我們可以用HCC病人開刀後的檢體所做成石蠟切片，利用FISH的方法來觀察其large ncRNA在肝臟組織細胞中的表現，此方法不但可以利用多年前的蠟塊檢體，也同時可觀察large ncRNA在細胞的分布情形，這是即時定量聚合酶連鎖反應所缺乏的。

關鍵字

肝癌

並列摘要

In human genome，only about 2% of nucleotides can encode proteins，while over 90% of the genome have non-protein coding transcripts (called non-coding RNA)。 In spite of much study focusing on miRNA, rRNA and tRNA，most of ncRNA have rarely been studied。 The cancer-related studies of ncRNA，excluding miRNA and a minor part of ncRNA，are also rarely studied， especially for the role of the very long ncRNA (over 5kb) in the development of cancers。The aim of this study is to use fluorescence in situ hybridization(FISH) method to analyze and compare the expression of ncRNA between Hepatocellular carcinoma (HCC) cancer and non-cancer cells。
In this experiment，the bioinformatic method was used to get 40 large ncRNA（over 5kb）. Using Real-time quantitative polymerase chain reaction (Real-time quantitative PCR) to analyze the expression of these large ncRNA in HCC. The result showed that the expression of HCC cancer cells was different from that of their neighbor normal cells。
The results were further confirmed using formalin fixed tissue and stain with fluorescence in situ hybridization(FISH)。 We therefore can use the paraffin section obtained from the HCC patient to observe the expression of large ncRNA in the liver tissue。Not only this method can use many years old the paraffin block , also it can observe large ncRNA in the cell's distribution, and it can not be achieved by Real-time quantitative PCR .

並列關鍵字

FISH ； Large Non-coding RNA

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