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研究生:駱逸涵
研究生(外文):Yi-HanLo
論文名稱:轉錄因子KLF10調節dnmt1基因的轉錄與肺癌惡化的關係
論文名稱(外文):Transcriptional regulation of dnmt1 gene by KLF10 associated with tumor malignancy of lung cancer
指導教授:王建平王建平引用關係
指導教授(外文):Jiang-Ping Wang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:67
中文關鍵詞:轉錄肺癌
外文關鍵詞:KLF10DNMT1
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根據衛生署所公布的九八年度國人十大死因,肺癌位居國人癌症死亡率之首,而其中癌細胞的轉移是造成肺癌病患存活率降低的主因,因此針對癌細胞轉移的分子機制之研究,對於肺癌病患的治療上佔有重要的地位。DNA methyltransferase 1(DNMT1) 蛋白質的主要作用是催化DNA進行甲基化修飾,先前研究指出DNMT1蛋白質會在癌細胞中大量表現,並與腫瘤的生成及轉移有著很大的關聯性;在許多癌細胞裡發現,腫瘤抑制基因上的啟動子都具有高度的甲基化修飾,點出了DNMT1的大量表現以及高度酵素活性,可能造成腫瘤抑制基因的不表現,因而促進腫瘤細胞的生成與轉移。在本篇研究中,我們發現轉錄因子Krüppel-like factor 10 (KLF10)會結合dnmt1啟動子上的SP1結合序列(SP1 binding site),抑制基因的轉錄。當我們利用誘導或轉染的方式,使KLF10蛋白質在非小細胞肺癌細胞中大量表現,會減低細胞中DNMT1的含量,並進一步地抑制癌細胞的增生與爬行。除此之外,我們還發現KLF10會與Specificity protein 1(SP1)競爭以調節DNMT1的表現量;已知SP1在癌細胞中具有高度表現量,並能結合dnmt1啟動子上的SP1結合序列,促進基因的轉錄,從我們的實驗中發現,當減低細胞中SP1的含量時,可以提升KLF10抑制DNMT1表現的能力,因此推測KLF10與SP1會競爭結合dnmt1啟動子上的SP1結合序列,調控dnmt1基因的轉錄。故在本篇研究中,我們發現KLF10會扮演抑制肺癌惡化的角色,並可以藉由調控DNMT1的表現量,來抑制肺癌細胞的生長與轉移,此外,我們也認為這個負向調控機轉可做為日後肺癌治療的參考。
Lung cancer is the leading cancer-related death in Taiwan. The major cause accounting for this severe disease is merely contributed by development of distal metastasis in its disease progression. Hence, it is important to investigate the underlying mechanism(s) of tumor metastasis for the therapeutic purpose of lung cancer. DNA methyltransferase 1 (DNMT1), a member of DNMT family, catalyzes the methylation processes of genomic DNA in somatic cells. Many studies have reported that overexpression of DNMT1 is found in a variety of cancers and associated with their tumorigenesis and tumor metastasis. Its well-known pathological function is involved in silence or attenuation of the transcriptional activation of many tumor suppressor genes (TSGs) by hypermethylation on their promoters. However, it is completely obscure how the DNMT1 gene expression is regulated and what nuclear factors(s) modulates its gene activation. In the Chip-chip analysis and the present study, Krüppel-like factor 10 (KLF10), dominantly expressed in G1 phase, was found to be a putative nuclear factor binding to the Sp1 binding sites on the dnmt1 promoter, and statistically downregulated in lung cancer tissues in immunohistochemistry study. Experiments using transiently or stably modulated the expression status of DNMT1 showed that KLF10 was an upstream negative regulator of its gene activation in non-small cell lung cancer cells. Moreover, suppression of cell proliferation and mobility by KLF10 was primarily mediated by regulation of its downstream target, DNMT1, gene expression. Increased expression of Sp1 markedly upregulated its gene expression. Consistently, attenuation of endogenous Sp1 by its gene-specific siRNA significantly decreased its expression. Interestingly, overexpression of KLF10 could further enhance the downregulated effect on dnmt1 transcriptional activity mediated by attenuation of Sp1, suggesting that KLF10 may regulate the transcription activity of Sp1. Collectively, the present study discloses that KLF10 is a novel negative regulator of the dnmt1 gene, suppresses DNMT1-medaited cell proliferation and cell migration, as well as modulates Sp1-mediated dnmt1 gene activation. Thus, KLF10 can be potential diagnostic and/or prognostic biomarkers for lung cancer.
目錄
中文摘----------------------------------------------------------------------------------- i
英文摘要------------------------------------------------------------------------------- ii
謝誌------------------------------------------------------------------------------------ iii
目錄------------------------------------------------------------------------------------- iv
圖目錄----------------------------------------------------------------------------------vi

前言--------------------------------------------------------------------------------------1
材料與方法
1. 細胞培養-----------------------------------------------------------------------------------------8
2. 基因轉染技術-----------------------------------------------------------------------------------8
3. 建立CL1-0 Tet-on KLF10 inducible stable clone細胞株-----------------------------8
4. 細胞株RNA萃取與純化---------------------------------------------------------------------9
5. 反轉錄聚合酶鏈鎖反應(RT-PCR)----------------------------------------------------------9
6. 西方墨點法(Western blot)------------------------------------------------------------------10
7. DNMT1抑制劑5-aza-2-deoxycytidine處理肺癌細胞株-----------------------------11
8. RNA干擾技術(siRNA)----------------------------------------------------------------------12
9. 細胞生長速率分析----------------------------------------------------------------------------12
10. 體外細胞爬行力分析(in vitro migration assay)-----------------------------------------12
11. 免疫組織染色(Immunohistochemistry; IHC)---------------------------------------13
12. 利用Thymidine同步細胞週期-------------------------------------------------------------14
13. Propidium iodide (PI)染色偵測細胞週期-----------------------------------------------14
14. 染色質免疫沉澱技術(Chromatin Immunoprecipitation;ChIP)------------------15
15. 雙色冷光酵素活性的測定(Dual-Luciferase assay)------------------------------------16
實驗結果
1. KLF10蛋白質會在正常肺泡組織中表現,然而在肺腺癌組織中卻會大幅度的降低-------------------------------------------------------------------------------------------------17
2. KLF10蛋白質的表現量會在肺癌細胞中大幅度的降低------------------------------ 18
3. 轉錄因子KLF10會坐落在DNMT1的啟動子上,並抑制啟動子的活性---------18
4. 異位性大量表現KLF10會抑制肺癌細胞中DNMT1的表現------------------------19
5. 在肺癌細胞中誘導KLF10表現可以降低細胞中DNMT1的表現量---------------20
6. 異位性大量表現DNMT1或是降低細胞中DNMT1的表現量都不會對KLF10的表現量造成影響-------------------------------------------------------------------------------21
7. KLF10表現量會在細胞週期中的G1 phase達到最多------------------------------- 22
8. KLF10可以藉由調節DNMT1的表現量進而抑制肺癌細胞的增生與爬行----- 24
9. 降低細胞中SP1的含量可以提升KLF10調節DNMT1表現的能力--------------26

討論------------------------------------------------------------------------------------ 30
參考文獻------------------------------------------------------------------------------ 36

圖目錄
圖一、KLF10蛋白質在肺腺癌組織中的表現情形------------------------------------------ 43
圖二、內生性蛋白質KLF10在不同肺癌細胞以及正常細胞的表現情形-------------- 45
圖三、偵測KLF10與DNMT1啟動子的關係----------------------------------------------- 46
圖四、異位性大量表現KLF10在肺癌細胞中對內生性DNMT1的影響------------- 48
圖五、誘導KLF10在細胞中大量表現對內生性DNMT1的影響----------------------- 50
圖六、改變細胞中DNMT1的表現量對內生性KLF10的影響-------------------------- 51
圖七、利用細胞飢餓法同步細胞週期---------------------------------------------------------- 52
圖八、觀察NHBE細胞中的KLF10在細胞週裡的表現量變化情形------------------- 53
圖九、觀察CL1-0細胞中的KLF10在細胞週期裡的表現量變化情形---------------- 55
圖十、高濃度的血清刺激細胞對KLF10的影響-------------------------------------------- 57
圖十一、利用Thymidine同步CL1-0的細胞週期並觀察KLF10在細胞週期中的表現量變化情形-------------------------------------------------------------------------------------------58
圖十二、誘導KLF10在肺癌細胞中穩定表現對癌細胞生長的影響-------------------- 59
圖十三、誘導KLF10在肺癌細胞中穩定表現對癌細胞爬行能力的影響-------------- 60
圖十四、破壞KLF10對DNMT1的負向調控機制後,對癌細胞爬行能力的影響- 62
圖十五、SP1蛋白質在不同肺癌細胞以及正常細胞的表現量----------------------------- 63
圖十六、異位性大量表現SP1或是減低癌細胞中SP1蛋白質的含量對DNMT1的影響------------------------------------------------------------------------------------------------------- 64
圖十七、同時異位性表現KLF10和減低細胞中SP1的含量對細胞中DNMT1
影響---------------------------------------------------------------------------------------------------- 65

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