論文名稱:

口腔鱗狀上皮癌細胞類胰島素生長因子結合蛋白-5啟動子的調控

 

Regulation of Insulin-like Growth Factor Binding Protein-5 Promoter in Oral Squamous Cell Carcinoma

研究生:

侯崇平  Chung-Ping Hou

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

林姝君  Shu-Chun Lin

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

39217005

          學年度:

93

          語文別:

中文

          出版年:

94

關鍵字:

類胰島素結合蛋白-5  IGFBP-5

 

薑黃素  curcumin

 

5-氮-2’-去氧胞嘧啶  5-aza-dC

 

亞硫酸基因定序分析  Bisulfate genomic sequencing

 

甲基化  mathylation

全文說明:

(本論文 20070822 對校內公開)

 

電子全文

        論文頁數:

86

摘要:

類胰島素生長因子結合蛋白(insulin-like growth factor binding proteinsIGFBPs)的家族成員眾多,而這些結合蛋白可與IGFs結合以達到抑制IGFsIGF接受器結合的能力,進而調控了IGFs促進細胞分裂的功能。在過去之研究發現,IGFBP-5具抑制腫瘤生長的功能。本實驗室先前的研究結果也觀察到,大部分的口腔鱗狀上皮癌細胞株中,不論在蛋白質層級或mRNA層級皆無法偵測到IGFBP-5的表現,然而在多株正常的口腔角質細胞中卻一致性地有表現IGFBP-5且與分化有關;另外薑黃素(curcumin)可調升SAS細胞株及正常口腔角質細胞之IGFBP-5 mRNA與蛋白質層級的表現量。但是造成口腔癌細胞株IGFBP-5表現調降,以及薑黃素調升IGFBP-5表現的原因,為本研究探討之課題。

 

本研究針對IGFBP-5啟動子的調控探討,(1)口腔鱗狀上皮癌細胞株之IGFBP-5基因不表現是否因其基因高度甲基(hypermethylation)化所致。以亞硫酸鈉修飾口腔鱗狀上皮癌細胞株的基因體DNA再加以定序,結果發現OECM-1HaCaT細胞株IGFBP-5基因不表現的原因與其基因第一外子附近的CpG島位密集區之甲基化程度有關。亦發現利用5-aza-dCDNA去甲基化後可提升IGFBP-5啟動子的活性,間接地使OECM-1HaCaT細胞株回復IGFBP-5 mRNA的表現量。(2)探討薑黃素是否會透過調控IGFBP-5啟動子的活性進而提升IGFBP-5 mRNA與蛋白層級的表現。以啟動子活性分析實驗結果發現薑黃素可以增加IGFBP-5啟動子活性,且呈現劑量依附性;透過刪除定譜分析(deletion mapping)深入分析發現薑黃素調控IGFBP-5啟動子活性的關鍵位置落在IGFBP-5基因序列轉錄起點算起-84+23的區域,此區域富含重要轉錄因子結合位置,是為探討IGFBP-5在口腔上皮細胞轉錄調控之重要基礎。

   
 

Insulin-like growth factor binding proteins is a family of protein that can bind IGF. They might exert inhibitory or enhancive effects on IGF-IGF receptor binding, that regulates cellular phenotypes. Previous studies have shown that IGFBP-5 is suppressive for tumor growth. Our previous study also indicated the profound down-regulation of IGFBP-5 mRNA and protein expression in oral carcinomas. Interestingly, IGFBP-5 is expressed in normal oral keratinocyte that is tightly associated with differentiation. Curcumin can up-regulate the IGFBP-5 mRNA and protein expression in SAS oral carcinoma cell line and normal oral keratinocyte. However, by what regulatory mechanisms that IGFBP-5 is down-regulated in OSCC and up-regulated by curcumin in oral epithelial cells are the main topics to be addressed in this study.

 

Using bi-sulfite modification and sequencing analysis, we identified that the intensity of CpG islands hypermethylation in exon 1 could underlies the silencing IGFBP-5 in OECM-1 oral carcinoma cell and HaCaT cells. Treatment with 5-aza-dC retrieved the IGFBP-5 mRNA expression in these cells. Such treatment also induced the promoter activity of IGFBP-5. It is hitherto unclear that the up-regulation of IGFBP-5 is mediated through a cis- or trans- activation. By promoter activity assay, we identified that curcumin can activate IGFBP-5 promoter in a dose-dependent manner. Deletion mapping further dissected that -84 ~ +23 in IGFBP-5 is the critical segment responsible for curcumin effects. Since this region encompassing multiple transcription factor binding sites, it will be served as a target to further elucidate the regulation of IGFBP-5 in oral epithelial cells.