Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21572
標題: PCDH21基因於乳癌檢體及乳癌細胞株之基因甲基化修飾調控及其功能性分析
The protocadherin γA-12(PCDH21) promoter hypermethylation and gene function studies in sporadic breast tumors and breast cancer cell lines
作者: 江敏瑜
Chiang, Ming-Yu
關鍵字: Protocadherin γA-12(PCDH21)
甲基化晶片
MS-PCR
morhpologic change
wound healing assay
metastasis
Protocadherin γA-12(PCDH21)
接觸性抑制作用
轉染試驗
癒合試驗
細胞生長速率
出版社: 生命科學系
摘要: 鈣黏附蛋白分子是一群表現於細胞膜,擔責細胞與細胞吸附作用的穿膜醣蛋白,在腫瘤發生的過程中對維持細胞型態、細胞接觸性抑制作用和傳遞訊息扮演重要的角色。本實驗室於2003年利用甲基化晶片(CpG islands microarray)研究,觀察到一鈣黏附蛋白家族分子稱之protocadherin γA-12(PCDH21),其在乳癌檢體當中具高比例甲基化修飾異常現象。本次試驗即利用MS-PCR偵測PCDH21基因於73對乳癌檢體和4株乳癌細胞株之基因調控區與第一表現子區之甲基化狀態,結果顯示高達近90%的乳癌檢體於PCDH21基因調控區與第一表現子區呈現異常甲基化修飾; 4株乳癌細胞株(MCF7、T47D、MDA-MB-231、MDA-MB-468)中則高達100%呈現異常甲基化修飾狀態,此高比例異常甲基化修飾促使我們更進一步以乳癌細胞株進行PCDH21基因之功能性分析。 首先評估不同惡化程度之乳癌細胞株(MCF7、T47D、MDA231)其PCDH21 mRNA表現量,結果顯示低度轉移能力的MCF7具較高表現量,而高度轉移能力的MDA231則具較低表現量; 接續以去甲基藥物(5-Aza-dC)處理PCDH21基因高度甲基化之MDA231乳癌細胞株,其基因表現量於5-Aza-dC處理後呈現上升趨勢,綜合基因表現量與去甲基處理分析結果,選用MDA231乳癌細胞株進行PCDH21真核表現載體之細胞轉染試驗。轉染試驗結果顯示重新大量表現PCDH21於MDA231,其細胞形態由纖維母細胞形態轉型為上皮細胞形態,此為抑制癌細胞之上皮細胞間-葉細胞轉變(epithelial-mesenchymal transition, EMT)現象; 於細胞傷口癒合試驗與細胞生長速率分析中證實,MDA231之高度轉移能力與細胞增生能力亦明顯受抑制。 根據上述試驗結果推測,乳癌生成過程中PCDH21基因調控區與第一表現子區之異常甲基化修飾導致其基因靜默化,此為乳癌細胞呈現高度轉移而促使乳癌進一步惡化的原因之一。
The cadherins are a family of cell surface glycoproteins respon- sible for cell adhesion, play an important role in cell morphology, contact inhibition and signal transduction during tumorgenesis. In our previous study, the PCDH21 hypermethylation was first found and identified in sporadic breast tumorsby using CpG islands microarray and methylation tissue amplicon array. In this study, we examined the methylation status of the PCDH21 promoter in breast cancers and breast cancer cell lines using methylation-specific PCR. Hypermethyl- ation of the PCDH21 in promoter and exon1 regions were high frequentl -y detected inTaiwan sporadic breast cancers with 92%(67/73) and 86(63/73),respectively. PCDH21 promoter aberrant methylation was dete- cted in 3 breast cancer cell lines, incliding MCF7, T47D,MDA468 and MDA231, with 100%.The highly aberrant methylation of PCDH21 in the breast cancer cells may imply its association with tumorigenesis. Thus, we further analyzed the function of PCDH21 in breaset cancer cell lines. Firstly, we evaluated the mRNA levels of PCDH21 in 3 breast cancer cell lines, including MCF7, T47D and MDA231, which represent different grade of malignancy. The lowest PCDH21 mRNA expression was observed in high metastasis MDA231, but the highest PCDH21 mRNA expression in low metastasis MCF7. The transcription regulation of the PCDH21 was demonstrated that directly controlled by DNA methylation modification using 5-Aza-dC demethylation agent treatment in MDA231 breast cancer cell line. In addition, overexp- ression of PCDH21 in MDA231 caused morphological changes that cells was switched from fibroblast into epithelial-like cells. The results of wound healing assay and cell growth rates assay both indicated that metastasis and proliferation of MDA231 were suppressed in the PCDH21-overexpression MDA231. Overexpression of PCDH21 in highly metastasis MDA231 would inducer morphologicalchanges, decrease cell migration ability and growth rate suggested that PCDH21 function as a potential tumor suppressor gene in breast cancer cell lines.
URI: http://hdl.handle.net/11455/21572
Appears in Collections:生命科學系所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.