- 學位論文
探討STAT3不同轉錄後修飾對於肺癌癌幹細胞特性的調控
Identifying the Independent Contributions of Three Canonical Posttranslational Modifications of STAT3 in Regulating Lung Cancer Stemness
摘要
信號傳導及轉錄激活蛋白3(Signal transducer and activator of transcription 3, STAT3) 屬於轉錄蛋白STAT家族的其中一員，受STAT3所調控之下游基因與細胞生長、分化、癌細胞轉移等特性息息相關；典型的激活模型指出當受到如上皮生長因子(EGFR)、介白質6 (Interlukin, IL-6) 等刺激時，STAT3會發生轉錄後修飾並聚合形成二聚體或是多聚體，進入細胞核與基因的啟動子區段結合，進而調控該基因表現；位於不同區段的轉錄後修飾會直接影響STAT3結構，並對STAT3的功能有所改變，此篇研究我們將討論酪胺酸705 (Tyrosine 705) 磷酸化、絲氨酸727 (Serine 727) 磷酸化、離胺酸685 (Lysine 685) 乙醯化在個別發生時對於肺癌癌幹細胞特性的調控，我們透過常間回文重複序列叢集/常間回文重複序列叢集關聯蛋白（Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-Associated Protein 9, CRISPR/ Cas9）基因編輯系統將STAT3剔除，並轉染具有兩個位置或三個位置顯性抑制性突變的STAT3，並針對STAT3的入核情形以及下游所調控的基因作探討。實驗結果顯示無論在兩個位置或是三個位置被顯示抑制突變的情形下，細胞核的區域皆能偵測到STAT3的訊號，此結果證明了其他修飾點位對於STAT3入核也有所影響；而針對與EMT相關之Twist1調控，則發現在離胺酸685可被正常乙醯化的STAT3組別中，Twist1表現量與野生型(wild-type) STAT3並無顯著差異，顯示離胺酸685此點位對於STAT3調控Twist1的重要性。
並列摘要
Signal transducer and activator of transcription 3 (STAT3) is a well-known transcription factor in regulating genes related to cancer stemness. The canonical activation model of STAT3 suggests the importance of posttranslational modification (PTM), dimer formation, and nuclear translocation of STAT3 upon mediating the gene expression. Here, we aim to identify the independent contribution of three PTMs, including phosphorylation on tyrosine 705 (pY705) and serine 727 (pS727), and acetylation on lysine 685 (acetyl-K685), during STAT3 activation. First, STAT3-null lung cancer cells are generated by CRISPR-Cas9 knockout system, and expression of epithelial-mesenchymal transition (EMT)-related genes (N-cadherin, Twist) and stemness-related genes (Oct4, ABCB1) are found to be down-regulated after deletion of STAT3. Next, we introduce STAT3 mutants with double (Y705F/K685R, S727A/K685R, Y705F/S727A) or triple (Y705F/S727A/K685R) dominant-negative residue replacement. Although the capability of STAT3 nuclear translocation is not influenced when impairing S727, Y705, and K685 of STAT3, RT-PCR results show that acetyl-K685 STAT3 alone up-regulates the expression level of Twist1 equalling to the wild type STAT3. This study not only implies the potential of other PTMs in STAT3 dynamics but the specific role of acetyl-K685 in STAT3-dependent Twist1 regulation.