利用 SAGE 方法探討 IKKα 所調控基因之表現

Abstract

Nuclear factor - κB ( NFκB )是發炎與免疫反應的重要調控因子，亦連接發炎與癌症。NFκB 之活化主要經由IKK 磷酸化IκBα，導致其受分解， 進而使NFκB 進入核內活化下游之基因。IKKα 與IKKβ具高度結構之相似性， 在IKKα-/-與IKKβ-/-基因剔除的老鼠， 表現型( phenotype ) 之差異顯示兩者具有不同功能。IKKα可進入核內磷酸化Histone H 3 或CBP。本論文主要欲探討IKKα可調控哪些基因的表現， 因此利用SAGE ( Serial Analysis of Gene Expression )方式研究IKKwt 與IKKα-/- M E F 間基因的變化。我們取得2 3 0 0 0 個SAGE tag 並利用SAGE Genie 網站分析，有1 3 5 個基因具顯著差異。由RT-PCR 確認1 5 個基因， 8 0 % 符合SAGE 之結果； 部分基因亦受p65 或IKKγ 之調控。在分析過程中， 我們也找到一個未知基因之表現受IKK α 調控。進一步利用IPA ( Ingenuity Pathway Analysis )網站分析整體基因的變化， 比對這些基因與細胞功能或疾病的關係， 我們推測IKKα-/-會減少骨骼發育相關基因之表現， 並期盼能藉由研究這些基因之變化與IKKα 的關係，找出與疾病相關的治療方針。

NFκB acts as a central coordinator of immune and inflammatory responses. It is also a crucial mediator of inflammation-induced tumor growth and progression. NFκB activation depends on phosphorylation-induced proteolysis of the inhibitory IκB, which is mediated by IKK and allows NFκB to translocate to the nucleus and activate target genes. Although similarity exists between IKKα and IKKβ, gene knockout studies clearly demonstrate that IKKα and IKKβ are functionally different.IKKα has been found to regulate many signaling pathway independently of direct regulation of NFκB, for example the phosphorylation of Histone H3 and CBP. In this thesis, I attempt to investigate the gene expression profiles regulated by IKKα. Therefore, SAGE was performed to compare gene profiles between IKKwt and IKKα-/- MEF cells. We collected 23000 SAGE tags and analyzed by SAGE Genie website. 135 genes with significant differences between two pools of SAGE libraries were distinguished. Checking fifteen SAGE genes by RT-PCR, 80% match of the alteration was confirmed. Some genes were also regulated by p65 and IKKγ. We also detected a novel antisense-transcript which was up-regulated in IKKα-/- MEF.IPA (ingénuity pathway analysis) was used to correlate the expression profiles with functions. The results showed IKKα is probably related to skeletal development. We hope that new therapeutic targets can be identified by elucidating the relationship between IKKα and these networks.