利用全基因組核醣核酸干擾篩選技術鑑定人類肝細胞癌的放射線保護基因

Abstract

在台灣，肝細胞癌屬於盛行率第三名的惡性腫瘤。因為肝細胞癌的轉移率和術後腫瘤的復發率都相當高，使得肝細胞癌的預後非常差。放射線治療是一種普遍用來治療癌症的策略，利用高能量的放射線達到殺死癌細胞的目的。隨著放射線和影像診斷技術的進步，醫生可以直接透過立體定位的方式精準地將高能量放射線傳遞到腫瘤的位置。但是對於肝細胞癌，放射線治療的效果沒有比目前的主要治療肝細胞癌的方法更加有效，加上肝細胞對於放射線的容忍度低，且肝癌細胞和正常肝細胞的放射線敏感度差異性不高，使得放射性治療沒有歸納在肝細胞癌的正規治療方針中。為了改善放射線治療在肝細胞癌治療上的侷限性，以及找到有潛力的藥物標靶加強放射性治療對於肝細胞癌的療效，我們利用人類肝細胞癌細胞株Huh7及全基因組核醣核酸干擾篩選技術來鑑定人類肝細胞癌的放射線保護基因和放射線敏感基因(放射線保護基因: 當基因表現受到抑制時，可使細胞增加對放射線的敏感度; 放射線敏感基因: 基因表現受到抑制時，使細胞對放射線的抵抗性增加)。我們從實驗結果篩選到二十一個候選基因，包括了十六個放射線保護基因和五個放射線敏感基因。這些候選基因利用細胞群落形成分析的驗證後得到十四個放射線保護基因，當中又以Clusterin (CLU) 基因影響放射線敏感度的效果最為顯著。透過流式細胞儀分析細胞在經過放射線處理後細胞死亡的情形，除了更加確認CLU影響細胞放射線敏感度的角色，也發現此現象只專一地在游離放射線處理後出現。而在紫外線處理下CLU基因的抑制沒有明顯地促進細胞死亡，然而其詳細的功能與機制仍須日後進一步的探討。

Hepatocellular carcinoma (HCC), the third most prevalent malignant tumor in Taiwan, has a poor prognosis due to high rates of recurrence and metastasis. Radiotherapy is one of the modalities in cancer treatments. With the recent development of stereotactic radiosurgery technology, physicians can deliver precise doses of energy to an exact location (i.e. the tumor) and thus limit collateral damage to surrounding normal tissues. Despite such an advanced technology, radiotherapy has not yet been incorporated into standard management guidelines of HCC because of the unsatisfactory clinical outcomes. In order to improve the efficacy of radiotherapy in treating HCC, we carried out a genome-wide RNA-interference (RNAi) screen in Huh7 cells (a human HCC cell line) and identified many genes as radiation protector candidates (i.e. knockdown of such genes increases the sensitivity of Huh7 cells to a predetermined dose of radiation) which might be used as potential targets for drug design to enhance radiation sensitivity of HCC. We identified twenty-one candidates from the screening results, including sixteen radioprotectors and five radiosensitizers. All of the candidates were validated by colony formation assay. Among these candidates, clusterin is the most significant radio-protector. Here we demonstrated that suppression of clusterin expression enhances the radiation-mediated cell death. Furthermore, the phenomenon caused by clusterin suppression is specific for ionizing radiation, but not for ultraviolet (UV) radiation. However, the detailed mechanisms and functions of clusterin need to be further studied and elucidated in the future.