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研究生:陳遠生
研究生(外文):Yuan Sheng Chen
論文名稱:討論Histidine triad nucleotide binding protein 1在人類肝癌中扮演的角色
論文名稱(外文):Histidine triad nucleotide binding protein 1(HINT1), a theoretical tumor suppressor gene, loses its function in human hepatocellular carcinoma
指導教授:謝森永
指導教授(外文):S. Y. Hsieh
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:99
論文頁數:85
中文關鍵詞:肝癌細胞防衛機制規避細胞凋亡機制組氨酸三聚體核苷結合蛋白1
外文關鍵詞:hepatocellular carcinomahormesisapoptosis evasionHINT1
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肝癌是整個地球村內造成嚴重死亡的惡性腫瘤之一。分析癌症地圖,肝癌好發
於非洲及亞洲,在台灣也不例外。依本實驗室長期研究的對象主要利用低劑量之紫外光誘發癌細胞產生死亡及抵抗死亡的防衛機制(Hormesis)。藉由二維電泳分析找到42個表現量有差異的蛋白質。經由生物功能機制網路的重建,找出十一個與細胞凋亡有關的蛋白質。藉辨識出調控肝癌細胞死亡機制相關的生物功能途徑。其中找到促細胞凋亡蛋白質 Histidine triad nucleotide binding protein 1 (HINT 1), HINT1表現量在引起防衛機制後與一般的肝癌細胞有顯著差異。因抑制 HINT 1 表達會大大地增加肝癌細胞對紫外線的耐受性,我們因此認為 HINT 1 在肝癌細胞內可能具有抑制細胞癌化的特性。本研究擬進一步探討 HINT 1 在肝癌細胞是否扮演重要的抑制腫瘤形成的角色。本研究的主要目的為(1)利用small interfering RNA進行喪失促凋亡功能(Loss-of-pro-apoptotic function assays)的實驗,分析在肝癌細胞有否HINT1 喪失促細胞凋亡能力的證據;(2)檢測 HINT1 的表現量在人類肝癌細胞中是否有被抑制;(3)找出 HINT1 之 cDNA 是否有發生突變的情形,導致 HINT1 喪失其功能。從實驗結果顯示,在 30 及 70 mJ/cm2紫外線劑量下,抑制 HINT 1 表現可導致Mahlavu 及 Hep 3B 肝癌細胞株較對照組細胞株具有較高的存活率(p<0.05),但在SK-Hep 1、Hep G2、Tong 以及 Huh7 肝癌細胞株則無此現象,推測 可能HINT1功能的喪失有關,藉由定量聚合酶鏈鎖反應檢測肝癌病人檢體中HINT1之表現量,從47位肝癌病人檢體與5個正常肝組織比較 HINT1 之表現量,發現 HINT1在 21 % 的肝癌病人檢體中表達量二倍下降;50% 的肝癌病人檢體中表達量二倍上升;依西方墨點法檢測肝癌病人檢體中HINT1之表現量,23個肝癌病人檢體與2個正常肝組織比較有1.5倍差異分別為,44%的檢體中 HINT1 表現量下降,56%的檢體中 HINT1 表現量上升。利用鏈鎖反應及TA分子選殖的技術,分析 HINT1 之 cDNA的序列。結果顯示Mahlavu、SK-Hep1、Hep G2、Huh7以及Tong這五株細胞內並沒有任何基因缺失、重組或是錯義突變的情形;而在Hep 3B這株肝癌細胞株中,只發現一個無意義突變(nonsense mutation)。綜合以上實驗結果,HINT1 在 21% 的肝癌組織中導致表現量下降主要的原因可能是HINT1 在一些肝癌細胞喪失其促細胞凋亡功能的情形。此外從我們的定序結果並沒有找到任何導致 HINT1 喪失促細胞凋亡功能的突變。
Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and a leading cause of death in many countries. The epidemiology of HCC has marked demographic and geographic variations, occurring mainly in Africa and Asia.
The long-term object of studies in our lab is to investigate the molecules and pathways involved in regulation of apoptosis evasion in human hepatoma cells. We have used optimized UV to induce cellular hermetic responses, and identified both the proteins and pathways regulating cellular death(Hsieh et al., J Proteome Res 2009).
Of them, the Histidine triad nucleotide binding protein 1 (HINT 1) was of particularly interesting, since silencing of its expression endowed cells with resistance to
UV-induced cell apoptosis. Since apoptosis evasion is an essential process for carcinogenic tumor formation of cells, HINT1 is a candidate tumor suppressor gene for
human cancers inducing HCC. We thus hypothesized that HINT1 is a tumor suppressor gene playing significant roles in hepatocellular carcinogenesis.The aims of this study were
1) to examine whether there was any evidence showing loss of pro-apoptotic function of HINT1 in human HCC;2) to examine whether there was down-regulation of HINT1 in
HCC;3) to find any mutations that led to loss of HINT1’s function. First, we used XTT assay by ionic radiation compare cell sensitivity to apoptosis induction before and after silencing the expression of HINT 1. Our data showed that when HINT 1 had been silenced, HCC cells became more sensitive to apoptosis in Mahlavu and Hep 3B, but not
in SK-Hep 1、Hep G2、Tong and Huh7. Second, we used both quantitative polymerase chain reaction and immunoblotting assays to determine the expression level of patients.
Our data showed that HINT 1 was down-regulated in cases 50% and up-regulated HINT1 in 21% patients with hepatoma tissues compare to 5 normal liver tissues. We then
examined whether HINT1 loses its function by inactivation mutation at its mRNA. The entire coding sequences of HINT1 cDNA of six different HCC cell lines were cloned and
sequenced. We found 5 of them(Mahlavu、SK-Hep 1、Hep G2、Huh7 and Tong) had no mis-sense mutation. Only one no-sense mutation was found in Hep 3B cells.
We concluded that loss of pro-apoptotic function of HINT1 was found in some of the HCC cells. The functional loss of HINT1might be attributable to down-regulation of the
expression HINT1,though which was only found in approximate 21% of HCC. Our data did not suggest any mutation related to loss of proapoptotic function of HINT1 in HCC
cell lines.
目錄
指導教授推薦書
口試委員審定書
授權書…………………………………………………………………iii
誌謝……………………………………………………………………iv
縮寫表…………………………………………………………………v
中文摘要………………………………………………………………vi
英文摘要……………………………………………………………vii
目錄……………………………………………………………………viii
圖表目錄………………………………………………………………xi
表目錄…………………………………………………………………xiii
第一章
序論……………………………………………………………………1
一、 研究背景
1.1肝癌簡介…………………………………………………………1
1.2肝癌的形成………………………………………………………2
1.3肝癌細胞與細胞凋亡的關係……………………………………3
1.4細胞防衛機制(Hormesis)……………………………………5
1.5癌症基因的篩選…………………………………………………5
二、Histidine triad nucleotide binding protein 1(HINT 1)
………………………………………………………………………7
三、 實驗設計
……………………………………………………………………9
ix
第二章
材料與方法………………………………………………………………11
2.1 細胞株及細胞培養……………………………………………11
2.2 細胞轉染作用…………………………………………………11
2.3 si-HINT1抑制效果測試………………………………………12
2.4 定量聚合酶連鎖反應………………………………………12
2.5 紫外光照射實驗……………………………………………13
2.6 細胞存活實驗………………………………………………14
2.7 細胞蛋白之萃取……………………………………………14
2.8 蛋白濃度之測量……………………………………………15
2.9 西方墨點法………………………………………………… 15
2.10 肝癌細胞RNA萃取…………………………………………17
2.11 細胞mRNA之反轉錄………………………………………17
2.12 聚合酶連鎖反應……………………………………………18
2.13 PCR 產物純化………………………………………………19
2.14 TA clone……………………………………………………19
2.15 E. coli.中質體的轉形作用…………………………………20
2.16 E. coli. 質體的抽取…………………………………………20
2.17 序列取得及分析……………………………………………21
x
第三章
實驗結果………………………………………………………………22
3.1 實驗流程圖……………………………………………………22
3.2 HINT 1在各肝癌細胞株中的表現量………………………22
3.3 觀察肝癌細胞株silenced HINT1之效果……………………23
3.4 觀察肝癌細胞株silenced HINT1之後抵抗UV的存活率…23
3.5 HINT 1在病人檢體中的表現量……………………………24
3.6 確認藍白篩選後黏結上載體之序列正確性………………26
3.7 六株肝癌細胞株HINT 1定序結果…………………………26
第四章
討論……………………………………………………………………28
參考文獻………………………………………………………………33
附錄一…………………………………………………………………52
附錄二…………………………………………………………………54
xi
圖目錄
圖一、HINT 1 在各物種間之胺基酸序列比對圖……………………39
圖二、本研究所使用引子之位置圖……………………………………40
圖三、本實驗之實驗流程圖……………………………………………41
圖四、HINT 1 在六種肝癌細胞株間的表現量………………………42
圖五、利用定量聚合酶鏈鎖反應檢測轉染HINT 1 之默化RNA 的效
率………………………………………………………………………43
圖六、利用西方墨點法檢測肝癌細胞中轉染HINT 1之默化RNA 抑制
HINT 1 蛋白質量的效率……………………………………………44
圖七A、肝癌細胞於轉染HINT1 RNA 後抵抗UV 的存活率………45
圖七B、肝癌細胞於轉染HINT1 RNA 後抵抗UV 的存活率………46
圖八A、利用定量聚合酶鏈鎖反應檢測HINT1 在 47 個肝癌病人檢
體中的相對表現量……………………………………………………47
圖八B、利用定量聚合酶鏈鎖反應檢測HINT1 在 47 個肝癌病人檢
體中的相對表現量……………………………………………………48
圖九、用西方墨點法檢測HINT1 在肝癌病人檢體中腫瘤與非腫瘤組
織的相對表現量……………………………………………………49
圖十、篩選 HINT1 之 cDNA 序列是否正確黏於 T 載體之電泳膠
圖………………………………………………………………………50
xii
圖十一、比較六株肝癌細胞中HINT 1 之序列………………………51
xiii
表目錄
表一、Oligonucleotide Primers Used for Q-PCR and PCR……………37
表二、抗體清單…………………………………………………………37
表三、HINT 1 siRNA……………………………………………………38
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