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研究生:楊千慧
研究生(外文):Chien-Hui Yang
論文名稱:ATAT1在大腸直腸癌預後之應用
論文名稱(外文):Application of ATAT1 in predicting colorectal cancer prognosis
指導教授:林奇宏林奇宏引用關係
指導教授(外文):Chi-Hung Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:39
中文關鍵詞:大腸直腸癌預後指標
外文關鍵詞:ATAT1colorectal cancerprognosis
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微管可透過 α-tubulin acetyltransferase 1 (ATAT1) 將乙醯基轉移至 ɑ-微管蛋白的 Lys40 上,反之,去乙醯化則由 histone deacetylase 6 (HDAC6) 及 sirtuin 2 (SirT2) 進行。這三種酵素的作用結果,將調節整體 ɑ-微管的乙醯化程度。過去文獻中指出,這個乙醯化修飾會出現在較 "穩定" 的微管,當細胞進行爬行時,接近細胞前端的微管有較多的乙醯化修飾。此外,於乳癌和大腸癌,及乳癌細胞株的研究中,ATAT1 表現量或 ɑ-微管 Lys40 的乙醯化程度,在腫瘤組織及較具轉移能力的乳癌細胞株會有高度的表現。
此外 ATAT1 具有多種轉錄異構物,其 N 端為保留的乙醯基轉移核心,C 端分別具有不同序列。過去文獻鮮少探討不同轉錄異構物的功能差異,我們實驗室發現,不同轉錄異構物具有不同的分布,並可與不同蛋白質結合,並設計了一套 PCR 流程,可偵測不同異構物的相對表現量,可藉此探討不同 ATAT1 轉錄異構物是否可能具有不同功能。
在此,我將探討 ATAT1 或是乙醯化 ɑ-微管在大腸癌中能否成為好的診斷或預後指標。我們將第一至四期共 78 個大腸癌檢體,利用 qPCR 分析 ATAT1、HDAC6、SIRT2 基因表現量,結果發現,腫瘤組織中 ATAT1 隨著第一期到第四期有逐漸上升的趨勢,但在鄰近正常組織則呈現下降趨勢,並非如過去文獻中所述,腫瘤組織一定高於鄰近正常組織。但是精細分析第二期及第三期中,有復發和沒復發的檢體,則發現無法由 ATAT1 表現量加以鑑別;而第四期樣品中,原位腫瘤及轉移至肝臟的組織間,ATAT1 表現量也沒有明顯差異。
我進一步使用實驗室先前發展的 PCR 流程,分析 ATAT1 的異構物在組織間表現量的差異,初步篩選出幾種異構物在腫瘤的表現量高於鄰近正常組織,接著針對這幾組設計特定的 qPCR 引子進行精確分析,我們發現異構物 2N 與 4N 對腫瘤組織的鑑別力相對於整體 ATAT1 明顯提高。
最後,我想了解不同 ATAT1 異構物於癌症進程中扮演什麼角色。我利用 CRISPR/Cas9將轉譯終止碼換入大腸癌細胞株 HCT116 的 ATAT1 基因中,初步測試顯示,focal adhesion kinase (FAK) 的活化程度會下降,WNT 訊息傳導路徑中的基因表現會受影響,我再一一回補不同 ATAT1 異構物,確認是否不同現象只受特定 ATAT1 異構物調控。
Microtubule can be acetylated by α-tubulin acetyltransferase 1 (ATAT1) on K40 and the reverse reaction is catalyzed by histone deacetylase 6 (HDAC6) and sirtuin 2 (SirT2). The interaction of these three enzymes regulates the level of acetylated α-tubulin. Previous studies suggested that post translational acetylation often exists on “stable” microtubules which could explain that more acetylation toward the leading edge during cell migration. Otherwise, in the researches of colon and breast cancer, as well as breast cancer cell lines, the expression of ATAT1 and the acetylated α-tubulin were high in the tumor tissues and metastatic breast cancer cell lines.
In addition, ATAT1 has many kinds of transcript variants. The N-terminal of these variants is a conserved core region of acetyltransferase, but the functions of C-terminal divergent region were not studied well. In our recent studies, these variants have different distribution and could be associated with different proteins. We also developed a quick PCR method to determine the relative expression level of different variants.
In this study, I investigated whether ATAT1 or acetylated α-tubulin could be good prognostic or diagnostic markers in colon cancer. 78 cases of stage I to stage IV colon cancer samples were used to analyze their expression level of ATAT1, HDAC6, and SIRT2 by qPCR. The results showed that ATAT1 expression in the tumor tissues increased from stage I to stage IV but decreased in adjacent normal tissues. These were different from the previous study that reported the level of ATAT1 expression in tumors was always higher than in adjacent normal tissues. When I divided patients into non-recurrence and recurrence groups, I could not discriminate the samples by ATAT1 level. Moreover, there were no significant difference of ATAT1 level between in situ tumors and the metastatic tumors in stage IV samples.
Furthermore, I analyzed the expression of ATAT1 variants in clinical samples by using quick PCR developed in our lab. According to the results, I selected four variants whose expression in tumor was higher than in adjacent normal tissues. Next, I designed the specific qPCR primer sets to achieve precise quantification. I found that variants 2N and 4N had higher discrimination in identifying the tumor tissues when compared to the total ATAT1 level.
Above all, to investigate the role of different ATAT1 variants in cancer progression, I generated ATAT1 knockout HCT116 cells by using CRISPR/Cas9 system. The preliminary results indicated the activity of the focal adhesion kinase (FAK) was decreased and the WNT signaling pathway could be impaired. After restoring each ATAT1 variants respectively, whether specific variant is responsible for specific cellular function could be verified.
Table of Contents
致謝 i
中文摘要 ii
English Abstract iii
Table of Contents iv
Chapter 1 Introduction
1.1 Post translational modification of tubulin 1
1.2 Tubulin acetylation and the discovery of ATAT1 1
1.3 ATAT1 correlates to cancer 2
1.4 Multiple ATAT1 transcription variants expressed in human 2
1.5 Aim of this study 3
Chapter 2 Materials and Methods
2.1 Antibodies ---4
2.2 Clinical samples ---4
2.3 Plasmid construction and shRNA ---4
2.4 Cell culture ---4
2.5 Generation of ATAT1 knockdown, knockout and overexpression cells ---5
2.6 RNA extraction and cDNA synthesis ---6
2.7 PCR and quantitative real-time PCR ---6
2.8 Immunohistochemical staining and analysis ---7
2.9 Western blotting ---7
Chapter 3 Results and Figure
3.1 Patient summary ---8
3.2 ATAT1 mRNA level in 4 stages of colon cancer patients ---9
3.3 HDAC6 and SIRT2 mRNA level in 4 stages of colon cancer patients ---9
3.4 ATAT1, HDAC6 and SIRT2 mRNA level overall analysis --13
3.5 ATAT1, HDAC6A and SIRT2 mRNA level by different reference genes 14
3.6 Acetyltransferase level and deacetylase level in stage II 15
3.7 Ac-Tu level analyzed by IHC 16
3.8 ATAT1 transcription variants in colon cancer patients 17
3.9 Verification with specific qPCR primer of ATAT1 variants 20
3.10 -Generation of ATAT1 depleted human colon cancer cell line HCT116 22
Chapter 4 Discussion
4.1 ATAT1 expression in tumor versus normal 23
4.2 Housekeeping genes selected for quantitation of mRNA levels 24
4.3 Specific ATAT1 variants were involved in cancer progression 24
References ---------26
Appendix 1 ATAT1 locus from exon 1 ~ 3 ---------29
Appendix 2 CRISPR primer design 29
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