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研究生:許玉燕
研究生(外文):Yu-Yen Hsu
論文名稱:人類癌細胞中HREV107及RIG1基因表現與其甲基化相關性之研究
論文名稱(外文):Analysis of HREV107 and RIG1 gene expression and the association with methylation in human cancer cells
指導教授:姜淑媛
指導教授(外文):Shun-Yuan Jiang
學位類別:碩士
校院名稱:國防醫學院
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:2005
畢業學年度:93
語文別:中文
論文頁數:55
中文關鍵詞:甲基化
外文關鍵詞:methylation
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HREV107與RIG1是屬於HHREV107第二型腫瘤抑制基因家族的成員,這兩個基因廣泛表現於正常組織中,而在各種腫瘤細胞及某些原位腫瘤中會降低其表現。H-rev107或RIG1異常的表現會導致細胞生長受到抑制及促進細胞的凋亡與分化。啟動區異常的DNA甲基化,在腫瘤抑制基因失去活性的過程中扮演重要的作用機轉。許多基因在啟動區上包含CpG島,並且透過此機轉使得基因失去表現。小白鼠的H-rev107基因啟動區包含CpG島,且H-rev107降低表現是受到DNA高度甲基化所調控。人類HREV107是一具有CpG島的基因,而RIG1基因則不具CpG島。本論文是研究DNA甲基化在人類HREV107及RIG1基因表現上所扮演的角色與作用機轉。
利用半量化反轉錄聚合酶連鎖反應偵測到CC-M2與HL-60癌細胞中低量的HREV107表現。去甲基藥物5-AZA-2'-Deoxylcytidine (5-aza-dC) 可以增加此兩種細胞中HREV107的表現,並且隨者藥物劑量的增加其表現量亦隨之增加。透過甲基化專一性聚合酶連鎖反應、甲基化專一性序列分析及COBRA (combined bisulfite restriction analysis) 的實驗,印證5-aza-dC誘導HREV107表現是透過降低CpG島的甲基化。HL-60細胞中蛋白質合成抑制劑 (cyclohexamide, CHX) 之處理,並不會影響5-aza-dC恢復HREV107表現的情況。最後利用COBRA方法分析癌細胞中HREV107的表現與CpG甲基化的相關性。發現高度表現HREV107之CC-M1與HT-29細胞中,其HREV107基因CpG島中之CpG是低度甲基化。
5-aza-dC處理3至5天可以增加CC-M2與HL-60癌細胞中RIG1的mRNA表現量。然而利用甲基化專一性聚合酶連鎖反應的方式分析卻發現在CC-M2細胞中5-aza-dC並不影響RIG1啟動區甲基化的情況。此外,同時以5-aza-dC與CHX處理,並不會增加HL-60細胞中RIG1的表現,但是單獨處理CHX卻會增加2.6倍的RIG1表現量。
總結,在CC-M2與HL-60細胞中,5-aza-dC是透過不同的作用機轉調控HREV107與RIG1的表現。CpG島的高度甲基化在HREV107的低量表現佔有重要的角色。然而,RIG1的表現並非直接受到啟動區CpG島的甲基化所調控。
HREV107 and RIG1 belong to the family of HREV107 type II tumor suppressor gene. Both genes are ubiquitously expressed in normal tissues. Expression of the genes are down-regulated in cancer cell lines and primary tumors. Ectopic H-rev107 or RIG1 expression leads to inhibition of cellular growth and induction of cellular apoptosis and differentiation. Aberrant DNA methylation of the promoter region is a key mechanism for inactivation of genes that suppress tumorigenesis. Most genes silenced by this mechanism contain CpG island in the promoter. Promoter of the murine H-rev107 gene contains CpG island, and DNA hypermethylation within island lesds to the gene silence. A CpG island is identified in human HREV107, whereas it is absent in the RIG1 gene. This study investigated role and mechanism of DNA methylation on expression of human HREV107 and RIG1 genes.
Low levels of HREV107 expression was detected in CC-M2 and HL-60 cancer cells using the method of semi-quantitative reverse transcription polymerase chain reaction (PCR). The demethylation compound 5-AZA-2'-Deoxylcytidine (5-aza-dC) increased HREV107 mRNA levels in both cell lines in a dose dependent manner. The 5-aza-dC-mediated HREV107 induction was accompanied with a decrease in DNA methylation within the CpG island of HREV107, which was supported by methylation specific PCR, methylation specific DNA sequencing and combined bisulfite restriction analysis (COBRA). Also, treatment with the protein synthesis inhibitor cyclohexamide had no effect on 5-aza-dC induced HREV107 expression in HL-60 cells. Finally, the correlation between HREV107 expression and CpG methylation in cancer cells was analyzed using the COBRA method. CC-M1 and HT-29 cells that expressed the highest levels of HREV107 were the least methylated.
Similar to the effect on HREV107, 5-aza-dC treatment for 3 to 5 days induced RIG1 mRNA levels in CC-M2 and HL-60 cells. However, 5-aza-dC had no effect on methylation of RIG1 promoter in CC-M2 cells determined by methylation specific PCR. The 5-aza-dC-mediated increased in RIG1 mRNA was not effected by treatment with CHX, in contrast to 2.6-fold induced in RIG1 expression by treatment with CHX (2 μM) in HL-60 cells.
In conclusion, expression of HREV107 and RIG1 was regulated by 5-aza-dC at the epigenetic levels through different mechanism in CC-M2 and HL-60 cells. CpG island hypermethylation played a role in down-regulation of HREV107 mRNA levels. However, expression of RIG1 is not directly regulated by promoter CpG methylation.
正文目錄

………………………………………………………………………………….頁
第一章、緒言.............................................1
一、HREV107第二型腫瘤抑制基因家族..........................1
二、HREV107.............................................3
三、RIG1................................................5
四、A-C1................................................7
五、腫瘤抑制基因異常表現之機轉.............................8
六、本論文研究方向.......................................11
第二章、材料與方法.....................................................13
一、細胞培養............................................13
二、細胞中全核醣核酸 (total RNA) 的萃取...................14
三、半量化反轉錄聚合酶連鎖反應 (Reverse transcription polymerase chain reaction, RT-PCR) .....................................15
1、互補去氧核醣核酸 (cDNA) 的製備.........................15
2、半量化聚合酶連鎖反應 (polymerase chain reaction, PCR) .16
四、細胞核去氧核醣核酸 (Genomic DNA) 的萃取................17
五、Genomic DNA之Sodium Bisulfite處理....................18
六、甲基化專一性聚合酶連鎖反應 (Methylation specific PCR, MSP) ..19
七、HREV107基因CpG島基因片段載體之構築......................................20
1、製備CpG島基因片段.....................................................................20
2、加A反應 (A tailing) ......................................................................21
3、接合反應 (Ligation) ......................................................................21
八、轉型作用 (Transformation) .................................................................21
九、細菌質體製備及纯化...........................................................................22
十、COBRA (Combined Bisulfite Restriction Analysis) ............................23
第三章、結果.......................................................................................................26
一、人類大腸直腸癌與前骨髓細胞白血病細胞株之HREV107與RIG1基因表現之分析.................................................................................26
二、去甲基藥物5-aza-dC對癌細胞中HREV107基因表現的影響..........27
三、去甲基藥物5-aza-dC對癌細胞中RIG1基因表現的影響..................27
四、以MSP方法分析癌細胞中HREV107及RIG1基因啟動區甲基化與其相對之RNA的表現........................................................................28
五、蛋白質合成抑制劑CHX對5-aza-dC誘導之HREV107及RIG1基因表現的影響.....................................................................................30
六、以甲基化專一性核酸定序分析HL-60細胞中HREV107 CpG島之基因片段核酸序列.................................................................................30
七、以COBRA的方式分析比較不同癌細胞株之HREV107甲基化的程度.........................................................................................................31
第四章、討論.......................................................................................................33
第五章、參考文獻...............................................................................................38
圖目錄

…………………………………………………………………………………..頁
圖一、HREV107基因5’端CpG島位置之基因序列示意圖………………….45
圖二、RIG1基因之Exon及5’端CpG島位置之基因序列示意圖……………46
圖三、HREV107 CpG島基因片段載體之設計與構圖……………………….47
圖四、以半量化RT-PCR分析不同細胞中HREV107與RIG1基因的表現…..48
圖五、去甲基藥物5-aza-dC對大腸癌及前骨髓細胞白血病癌細胞HL-60中HREV107基因表現的影響……………………………………………………………………...49
圖六、去甲基藥物5-aza-dC對大腸癌及前骨髓細胞白血病癌細胞HL-60中RIG1基因表現的影響…………………………………………………………………………...50
圖七、以MSP方法分析癌細胞中HREV107及RIG1基因啟動區甲基化與其相對RNA表現之分析……………………………………………………………………………...51
圖八、蛋白質合成抑制劑CHX對5-aza-dC誘導之HREV107及RIG1基因表現的影響……………………………………………………………………………….52
圖九、分析3 μM 5-aza-dC處理3天對HL-60細胞HREV107基因中CpG島內核酸改變的情形……………………………………………………………………………….53
圖十、以COBRA的方法分析比較不同癌細胞株之HREV107基因CpG島基因片段的甲基化程度……………………………………………………………………………….54

表目錄

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