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研究生:陳俞均
研究生(外文):Yu-Chun Chen
論文名稱:DNA甲基轉移酶3A去甲基化抑制劑的合成藥物篩選平台的建立
論文名稱(外文):Set-up of Compound Screening Platform for Identification of DNMT3A Demethylation Inhibitors
指導教授:沈哲鯤沈哲鯤引用關係
指導教授(外文):Che-Kun James Shen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學系暨基因體科學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:44
中文關鍵詞:DNA甲基轉移酶去甲基化抑制劑藥物篩選平台
外文關鍵詞:DNA methyltransferasedemethylation inhibitorscreening platform
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去氧核醣核酸(DNA)的甲基化及去甲基化對於基因表現的調控十分重要。其中,負責去氧核醣核酸甲基化的酶被稱為去氧核醣核酸甲基轉移酶,它能夠將甲基從甲基提供者轉移到胞嘧啶的五號碳上。而去氧核醣核酸甲基轉移酶又可依作用偏好分為兩種類型,一種是在細胞複製時維持新股甲基化狀態和舊股相同的維持型去氧核醣核酸甲基轉移酶,像是去氧核醣核酸甲基轉移酶一型(DNMT1),另一種則是能夠建立新的甲基到胞嘧啶上的重生型甲基轉移酶,包含去氧核醣核酸甲基轉移酶三A型(DNMT3A)和三B型(DNMT3B)。另一方面,去甲基化路徑也分為兩種,分別是被動型及主動型,被動型去甲基化路徑不需要酵素協助,會在去氧核醣核酸進行複製時被動發生;而主動型路徑則需要酵素反應作用,首先胞嘧啶五號碳的甲基通過TET蛋白氧化,接著透過鹼基切除式修復(BER)或核苷酸切除式修復(NER)進行脫氨來完成。然而,我們實驗室發現重生型去氧核醣核酸甲基轉移酶,DNMT3A和DNMT3B在體外具有脫羥甲基化能力和去甲基化能力。在我們最新發表的文獻中,更證明了去甲基化能力也是能夠在細胞中看到的,另外我們更推測出小鼠的DNMT3B的去甲基化功能與甲基化活性位點可能共享了相同的剪切位。這一發現對於更理解胞內甲基化的動態平衡具有重大意義,為了進一步的研究,我們希望找到一些只能影響去甲基化能力但不影響甲基化能力的去氧核醣核酸甲基轉移酶抑製劑。因此,我們利用冷光素酶測定法作為材料,設計了細胞基礎的重生型去氧核醣核酸甲基轉移酶去甲基化抑製劑的藥物篩選平台,用以進行人類DNMT3A去甲基化抑制劑小分子藥物的超高速藥物篩選。另一方面,我們也建立了另一種以癌細胞為材料的細胞表型測試實驗,在篩選出一些候選藥物後能夠用來測試去氧核醣核酸甲基轉移酶的去甲基化是否會影響癌症進程。
DNA methylation and demethylation are critical for spatiotemporal modulation of gene expression. DNA methylation is generated by DNA methyltransferases, DNMTs, which can covalently add a methyl group to the 5-position carbon of cytosine (5mC). DNMTs are classified into two categories, namely, the maintenance and de novo enzymes. The maintenance enzyme DNMT1 is involved in maintaining methylation pattern during DNA replication, whereas the de novo enzymes DNMT3A and DNMT3B can add a new methyl group on the cytosine of CpGs. On the other hand, 5mC, which is methylated on the fifth carbon of cytosine, can be demethylated actively or passively. While passive demethylation results from DNA replication and does not require enzymatic activities, active demethylation of 5mC can take place following various enzymatic reactions. These include direct action of base excision repair and nucleotide excision repair; combined deamination reaction followed by action of base excision repair and, finally, oxidation of 5mC via TET family of DNA dioxygenases to sequentially generate 5-hydroxymethylacytosine, 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC). In the past from our lab, it has been shown that both of the de novo DNMTs, DNMT3A and DNMT3B possess dehydroxymethylation as well as demethylation activities in vitro. Recently, our lab showed that the demethylation activities of mouse DNMT3A and DNMT3B also exist in the cellular context. Furthermore, the demethylation activity of mouse DNMT3B might share the same catalytic site of its methylation activity. This finding is significant for understanding of the methylation homeostasis within the cells. In order to targeting the role of genome-wide demethylation in tumorigenesis, our lab has contended the possible involvement of DNA demethylation by the de novo DNMTs in this process and ventured into the designing of a high-throughput screening platform for the identification of small compounds that would inhibit this activity of the de novo DNMTs without affecting their methylation activity. For this, we have set up a cell-based luciferase reporter reactivation system, which we optimized to use as the screening platform. Since we would like to use the candidate in future, demethylation inhibitors for slowing down cancer progression, we have also established and optimized a series of cancer cell-based phenotypic assays for detecting thus verifying effects of the candidate inhibitors identified from screening.
Acknowledgments ------------------------------------------------------------------------------ i
Abstract ------------------------------------------------------------------------------------------ ii
摘要 --------------------------------------------------------------------------------------------- iv
Content -------------------------------------------------------------------------------------------vi
1. Introduction --------------------------------------------------------------------------------- 1
1.1 DNA methyltransferases --------------------------------------------------------------- 1
1.2 DNA methylation and cancer --------------------------------------------------------- 2
1.3 Mechanisms of DNA methylation and demethylation----------------------------- 4
1.4 Specific aim ----------------------------------------------------------------------------- 5
2. Material and method ------------------------------------------------------------------------ 7
2.1 Cell culture ------------------------------------------------------------------------------ 7
2.2 Reporter plasmid preparation --------------------------------------------------------- 7
2.3 In vitro methylation of plasmid DNA ----------------------------------------------- 8
2.4 SDS-PAGE and Western blot --------------------------------------------------------- 8
2.5 Forward transfection assay ------------------------------------------------------------ 9
2.6 Cell viability assay -------------------------------------------------------------------- 10
2.7 Cell migration assay ------------------------------------------------------------------ 10
2.8 Statistical analysis -------------------------------------------------------------------- 11
3. Result --------------------------------------------------------------------------------------- 12
3.1 The drug screening platform ------------------------------------------------------- 12
3.2 The cell phenotyping assay --------------------------------------------------------- 16
4. Discussion ---------------------------------------------------------------------------------- 19
5. Reference ----------------------------------------------------------------------------------- 22
6. Figure --------------------------------------------------------------------------------------- 29
Figure 1. A robust luciferase activity directed by the CMV promoter in comparison to the CPM promoter --------------------------------------------------------------------- 29
Figure 2. DNMT3A methylated activity in transfected HEK-293 cells ------------ 30
Figure 3. Cartoon chart showing the design of the screening platform ------------- 31
Figure 4. Confirmation of the DNA methylation by M. sssI. ------------------------ 32
Figure 5. Time course of DNA demethylation by DNMT3A ------------------------ 33
Figure 6. Two control experiments for the DNA transfection technique ----------- 34
Figure 7. Flow chart for preparation of transfected cells ----------------------------- 35
Figure 8. The reactivation effect assay by fresh cell protocol or frozen cell protocol ----------------------------------------------------------------------------------------------- 36
Figure 9. Flow chart representing the final protocol for screening ------------------ 37
Figure 10. Determination of cytotoxicity concentration 50% (CC50) of 5-aza-dC treatment ----------------------------------------------------------------------------------- 38
Figure 11. Migration assay of A549 cells treated with 5-aza-dC (DAC) ----------- 39
Figure 12. Cell viability assay of SW480 cells under hypoxia condition ---------- 40
Figure 13. Migration assay of SW480 cells in hypoxia condition ------------------- 41
7. Table ---------------------------------------------------------------------------------------- 42
Table 1. Comparison of the fresh and frozen cells in HTS facility ------------------ 42
Table 2. Compound tests in HTS facility ----------------------------------------------- 43
8. Supplementary figure --------------------------------------------------------------------- 44
Supplementary figure 1. Map of the commercial pGL3-control vector ------------ 44
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