IKK調控組蛋白甲基轉移酶G9a促進癌症發展之探討

IKK Regulates Histone Methyltransferase G9a to Promote Cancer Progression

李啟恆(Kai-Hang Lei)

指導教授 ： 郭明良

國立臺灣大學/生命科學院/生化科學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.00589

摘要

G9a 是哺乳動物的組蛋白甲基轉移酶，在不同癌症中常常會高度表現，使得抑癌基因靜默而無法轉錄，調控腫瘤發展的進程。由於轉譯後修飾對於調控蛋白的功能十分重要，而目前沒有文獻探討G9a的轉譯後修飾，因此我們利用生物資訊的軟體預測G9a可能的磷酸化位置，在其中發現可能具有IKKa/b的磷酸化位置。
在本篇的研究中，我們在293T細胞和乳癌細胞MCF-7、BT549中利用免疫沉澱方法以及GST pull down assay 證明了G9a可以在in vivo及 in vitro的情況下跟IKKa/b結合，在in vitro kinase assay的實驗中證明了IKKa/b確實可以磷酸化G9a。為了進一步探討IKKa/b對於G9a的調控，我們在293T cell過度表現IKKa/b後，發現G9a的蛋白質表現量會有明顯上升而RNA的量沒有太大改變，推斷IKKa/b可能影響到G9a轉譯或者是蛋白質的穩定度。因此在293T細胞中加入cycloheximide，可以發現過度表現IKKa/b的細胞可以減緩G9a的降解速度，而在MCF-7 細胞中把IKKa/b剔除則會使G9a的表現量下降，另外當加入細胞激素IL-1b後也可以看到G9a的蛋白質表現量有一定的上升，代表IKKa/b的活性調控G9a的穩定度。在乳癌細胞MCF-7中剔除IKKa/b也會造成細胞的生長、轉移和侵襲的能力下降，而由G9a所抑制的基因則是有所上升。由於G9a在癌症發生過程中扮演重要的調控角色，本篇研究發現IKKa/b可以藉由磷酸化G9a來調控它的功能來促進癌症發生，未來或許在G9a表現比較高的病人中，可以同時針對IKK跟G9a進行抑制，提高治療效果。

關鍵字

IKK ； 組蛋白甲基轉移酶 G9a

並列摘要

G9a, also known as EHMT2, is a histone H3 lysine 9 methyltransferase, has been observed overexpression in various human cancers and contributes to the epigenetic silencing of tumor suppressor genes to promote cancer progression. Post-translational modification (PTM) is very important to regulate protein function. It has been shown that several epigenetic regulators were regulated by different PTMs. The post translational modification that regulates function of G9a had not been reported yet, therefore it is necessary to investigate the PTM of G9a. Interestingly, from the bioinformatics prediction, we found that G9a contains IKKa/b consensus phosphorylation site, suggesting a possible interaction between IKKa/b and G9a.
In this study, we used the co-immunoprecipitation in 293T cells, MCF-7 and BT549 breast cancer cells and GST pull down assay to prove that G9a interacted with IKKa and b in vivo and in vitro. By using in vitro kinase assay, we proved that G9a can be phosphorylated by both IKKa and b. To investigate the regulation of G9a by IKKa/b, we overexpressed IKKa/b in 293T cells and found the protein expression level of G9a was increased but not the RNA level. It suggested that IKKa/b may regulate the translation activity or protein stability of G9a. For this reason, we added cycloheximide in 293T cells to examine the half-life of G9a. The cells with overexpression of IKKa/b can prolong the half-life of G9a, on the contrary, knockdown of IKKa/b decreased G9a protein level in MCF-7 cells. Furthermore, the cytokine IL-1b can also enhance the protein level of G9a in MCF-7 cells, suggesting that the kinase activity of IKKa/b regulated protein stability of G9a. Knockdown of IKKa/b in MCF-7 cells inhibited cell growth, decreased migration and invasion ability, and increased the RNA level of the genes that suppressed by G9a.
G9a had played an important role in cancer progression, this study reveals that IKKa/b can phosphorylate G9a to promote cancer progression. Targeting IKK and G9a in the cancer patients with G9a overexpression may potentially enhance the therapeutic efficacy in the future.

並列關鍵字

IKK ； histone methyltransferase G9a

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