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系統識別號 U0026-0812200915302875
論文名稱(中文) 在人類大腸癌細胞內建立一個微衛星不穩定偵測系統
論文名稱(英文) Development of a Microsatellite Instability Reporter System in Human Colorectal Cancer
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 陳伊婷
研究生(英文) Yi-ting Chen
電子信箱 t1696407@mail.ncku.edu.tw
學號 t1696407
學位類別 碩士
語文別 英文
論文頁數 85頁
口試委員 指導教授-張玲
口試委員-孫孝芳
口試委員-吳梨華
中文關鍵字 (CG)微衛星序列  DNA錯誤配對修復  微衛星不穩定  過氧化氫  大腸癌 
英文關鍵字 DNA mismatch repair  (CG)-repeats  Microsatellite instability  Colorectal cancer  H2O2 
學科別分類
中文摘要 大腸癌近幾年來已經攀升至台灣十大癌症死因的第三位。 遺傳性非息肉症大腸直腸癌患者,其DNA錯誤配對修復基因帶有遺傳性的缺陷,導致DNA錯誤配對修復機制的正常功能喪失。 另外,偶發性大腸癌病例當中,也發現hMLH1基因的啟動子過度甲基化,進而導致此DNA錯誤配對修復基因的功能喪失。 因此,不論是基因遺傳或表觀遺傳上的變異,都可能導致DNA錯誤配對修復機制無法正常運作。 同時,先前研究也發現氧化壓力可以使DNA錯誤配對修復機制失去校對能力。 當DNA錯誤配對修復機制無法正常校對時,則無法修復微衛星序列長度上在DNA複製時所產生的錯誤,導致微衛星不穩定性(MSI)。 MSI除了在大腸癌之外,也可在潰瘍性大腸炎患者中被偵測到。 潰瘍性大腸炎患者,罹患大腸癌的機率普遍比一般人高。 帶有MSI的病人(MSI+)也較容易對化療產生抗藥性。 為了要減少大腸癌的發生率和死亡率,預防MSI的發生和發展是很重要的。 既然大腸癌可以受到基因遺傳跟表觀遺傳變異的影響,我假設此微衛星不穩定的表現型可作為同時偵測基因遺傳和表觀遺傳變異的指標。 為了證明此假設,我建立了一套帶有(CG)5,(CG)8,(CG)13三種不同長度微衛星的雙螢光MSI報導系統。 在DNA錯誤配對修復機制帶有缺陷的人類大腸癌細胞株(HCT116)中,我建立了至少兩個各別可以穩定表達(CG)n微衛星序列以及(N)26隨機序列的大腸癌細胞。 在穩定表達(CG)n序列的細胞中,過氧化氫(H2O2)的劑量和微衛星不穩定的發生頻率(MSI frequency)成正比。 在不同序列長度的微衛星裡,又以(CG)13較(CG)5容易偵測並且報導細胞中MSI的表現型。 同時,MSI frequency也隨著H2O2處理的次數增加而增加。 透過直接定序帶雙色螢光的(MSI+)細胞內DNA,我發現(CG)13的微衛星序列少了一個(CG)單元。 此結果也間接說明了紅色螢光重新表現的原因。 然而,在缺乏葉酸的實驗中,至少在20天的實驗組中我並未發現MSI發生頻率和缺乏葉酸有直接且明顯的關聯性。 未來,我建立的MSI螢光報導系統可用於大量篩選抗微衛星不穩定的藥物(anti-MSI compounds),以期減少大腸癌的發生率和死亡率。
英文摘要 Colorectal Cancer (CRC) is ranked as the 3rd leading cause of cancer mortality in Taiwan. Patients with inflammatory disease such as ulcerative colitis have a high risk of colon cancer. Germline mutations in the DNA mismatch repair (MMR) genes account for hereditary nonpolyposis colorectal cancer (HNPCC). On the other hand, hypermethylation of the hMLH1 promoter accounts for sporadic colon cancer. Both genetic and epigenetic events lead to MMR inactivation. Additionally, oxidative stress also inactivates the function of MMR. Inactivated MMR is unable to correct insertions or deletions of the repeat unit in microsatellites, which is manifested as microsatellite instability (MSI). MSI has been detected in patients with colon cancer and ulcerative colitis, and MSI-positive patients will develop drug resistance. To reduce CRC incidence and mortality, it is important to prevent MSI from development. Since CRC is caused by both genetic and epigenetic alterations, we hypothesize that the MSI phenotype serves as a nexus to simultaneously detect epigenetic (such as DNA methylation) and genetic (such as insertions/deletions) events. I constructed a dual fluorescence MSI reporter system consisting of (CG)5, (CG)8 and (CG)13 microsatellites. From MMR-deficient HCT116 human colorectal cancer cell line, I generated at least two stable clones harboring each (CG)n microsatellite , as well as a negative control harboring the (N)26 random sequence. H2O2 increased the MSI frequency in a dose-dependent manner in the stable clones harboring (CG)n. Compared to (CG)5, (CG)13 appears to be more sensitive to report the MSI phenotype. Furthermore, H2O2 also increased the MSI frequency in a time-dependent manner. Direct sequencing of MSI-positive (i.e., RFP+/GFP+) cells revealed that one (CG) unit deletion was a major event that restored the correct reading frame of RFP. In the future, the MSI reporter system will be used for screening anti-MSI compounds in order to reduce the incidence and mortality of colorectal cancer.
論文目次 1. INTRODUCTION………………………………………………….9
1.1. Colorectal Cancer………………………………………………………….9
1.2. Microsatellites…………………………………………………………...10
1.3. Microsatellite Instability………………………………………………..11
1.4. Mismatch Repair System……………………………………………….13
1.5. Epigenetic alterations…………………………………………………...15
1.6. Folate and colorectal cancer……………………………………………16
1.7. Hypothesis……………………………………………………………….18
1.8. Specific Aims…………………………………………………………….19
2. MATERIALS AND METHODS…………………………………20
2.1. Reagents…………………………………………………………………20
2.2. Cell lines…………………………………………………………………21
2.3. Cell culture………………………………………………………………21
2.4. Cloning of Dual Fluorescence Reporter Plasmid……...……………...22
2.5. Transfection……………………………………………………………..25
2.6. Limiting Dilution……………………………………………………......25
2.7. MTT assay……………………………………………………………….26
2.8. H2O2 treatment………………………………………………………….27
2.9. Folate-deficient treatment……………………………………………...28
2.10. Flow cytometry……………………………………………………….….28
2.11. Bisulfite treatment……………………………………………………….29
2.12. Methylation Specific Polymerase Chain Reaction………………….…29
2.13. Microsatellite instability (MSI) assay………………………………..…30
2.14. Microsatellite sequence analysis of hMSH3 and hMSH6…………..….31
3. RESULTS………………………………………………………….33
3.1. Construction of the dual fluorescence MSI reporter plasmid containing the (CG)n microsatellites………………………………………………..33
3.2. The effect of H2O2 on the MSI frequency in transient transfectants derived from HCT116 and HCT116+ch3 human colorectal cancer cell lines………………………………………………………………………34
3.3. Generation of stable clones harboring dual fluorescence MSI reporters…………………………………………………………………35
3.4. The effect of H2O2 on the MSI frequency in HCT116-derived stable clones…………………………………………………………………….36
3.5. Analysis of the exogenous and endogenous microsatellites in the GFP+/RFP+ enriched subpopulation………………………………….37
3.6. The effect of folate on the MSI frequency in stHCT116-(CG)5 and the stHCT116-(CG)13 stable clones………………………………………...39
3.7. The effect of folate on the promoter methylation in human colorectal cancer cell lines…………………………………………………………40
4. DISCUSSION……………………………………………………...41
5. REFERENCES……………………………………………………44
6. TABLES……………………………………………………………50
Table 1. The microsatellite insert sequences…………………………………50
Table 2. The estimation on MSI detection limits…………………………….51
Table 3. The methylation status on the colorectal cancer cell lines…………52
Table 4. The primers and settings for Methylation Specific Polymerase Chain Reaction……………………………………………………….53
Table 5. The 5 NCI recommended markers for MSI assay…………………54
7. FIGURES……………………………………………………………55
Figure 1. Folate in one-carbon metabolism………………………………..55
Figure 2. A schematic presentation of the dual fluorescence MSI reporter constructs containing (CG)n (n=5, 8, 13) microsatellites………..56
Figure 3. Confirmation of the dual fluorescence MSI reporter constructs containing the (CG)5 microsatellite……………………………..57
Figure 4. Confirmation of the dual fluorescence MSI reporter constructs containing the (CG)8 microsatellite……………………………..58
Figure 5. Confirmation of the dual fluorescence MSI reporter constructs containing the (CG)13 microsatellite…………………………….59
Figure 6. Determination of LD50 of H2O2 in the HCT116 human colorectal cancer cell line…………………………………………………….60
Figure 7. Effects of H2O2 on transient transfectants derived from in HCT116 and HCT116+ch3 human colorectal cancer cell lines..61
Figure 8. Generation of HCT116-derived stable lines that harbor the dual fluorescence reporter containing the (CG)5 microsatellite……..63
Figure 9. The HCT116 stable clones harbored the dual fluorescence reporter containing the (CG)8 microsatellite…………………...66
Figure 10. The HCT116 stable clones harbored the dual fluorescence reporter containing the (CG)13 microsatellite……………..67
Figure 11. The effect of H2O2 on the stHCT116-(CG)5 stable cell lines….69
Figure 12. The effect of H2O2 on the stHCT116-(CG)8 stable cell lines…71
Figure 13. The effect of H2O2 on the stHCT116-(CG)13 stable cell lines…73
Figure 14. Comparison of H2O2 effect on HCT116-derived stable harboring (CG)5 and (CG)13 microsatellites…………………..77
Figure 15. Direct sequencing of the genomic DNA extracted from enriched GFP+/RFP+ or GFP+ subpopulation…………………………….79
Figure 16. Analysis of the (A)8 microsatellite in the hMSH3 coding region in human colorectal cancer cell lines and derivatives…………...80
Figure17. Analysis of the (C)8 microsatellite in the hMSH6 coding region in human colorectal cancer cell lines and derivatives…………...82
Figure 18. Analysis of the methylation status of human colorectal cancer cell lines…………………………………………………………….84
Figure 19. The effect of folate on the MSI frequency in stHCT116-(CG)5 and stHCT116-(CG)13 stable lines……………………………………..85
Figure 20. Folate deficient effect on the p16 promoter methylation………..86
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