Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92244
標題: Study of the role of YWHAZ in lung cancer and identification of its downstream genes
探討YWHAZ蛋白在肺癌中扮演的角色及其調控下游基因
作者: 蔣依庭
I-Ting Chiang
關鍵字: 肺癌;YWHAZ;LUNG CANCER
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摘要: 
YWHAZ, also known as 14-3-3ζ, belongs to the 14-3-3 family of proteins which mediate signal transduction by binding to phosphoserine-containing proteins. It's believed that YWHAZ plays an important role in cell signaling, cell cycle, and apoptotic death. YWHAZ is one of the major transforming growth factor-β-induced proteins, which can promote epithelial–mesenchymal transition of epithelial cells in cancer cell transformation. In our previous studies, we found that YWHAZ associates with TCF-4 and β-catenin in the nucleus and further promote EMT, cell invasion and migration by activating the YWHAZ/β-catenin axis in lung cancer. In order to investigate downstream genes of YWHAZ in lung cancer, Microarray and ChIP-seq assay were used to select candidate genes which may be regulated by YWHAZ. First, the genes that were up- or down-regulated by YWHAZ more than 1.5-fold and simultaneously contain TCF-4 binding motif in their 5'-upstream regulatory region determined by ChIP-seq were identified. We obtained fourteen candidate genes that might be regulated by YWHAZ. Next, we found that mRNA expression of nine candidate genes were regulated by knockdown of YWHAZ with siRNA or short hairpin RNA (shRNA) and recovered by re-expression of YWHAZ in the stable clones that harbor shRNA in CL1-5 cell line. Moreover, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between the YWHAZ-β-catenin-TCF-4 complex and four candidate genes: ATF3, DMTF1, DUSP1 and PAK2. Electrophoretic mobility shift assay (EMSA) was performed that protein recognized and bound the TCF-4 binding motif on DNA of the promoter of four candidate genes. Taken together, our data reveal that YWHAZ can regulate these candidate genes through forming the regulatory complex with TCF-4 and β-catenin.

YWHAZ蛋白,又稱為14-3-3ζ,屬於14-3-3蛋白家族的一員。14-3-3蛋白家族會藉由與含有磷酸化serine的蛋白(phosphoserine-containig protein)結合,來調控細胞的訊號傳遞(signal transduction)與生理功能。YWHAZ蛋白又為轉型生長因子β誘導蛋白(transforming growth factor-β-induced protein)之一,可促進癌細胞上皮細胞轉型間葉細胞過程(epithelial-mesenchymal transition;EMT)的發生,促使癌轉移的產生。在實驗室先前的研究發現,YWHAZ蛋白會與β-catenin蛋白和轉錄因子T-cell factor 4 (TCF4)在核中結合,形成一複合體(complex),並促進EMT、癌細胞侵襲(invasion)和移動(migration)的發生。為了進一步研究在肺癌細胞中是否有其他基因受YWHAZ所調控,進而促進肺癌進程(lung cancer progression)的發生,我們利用微陣列分析(microarray)和染色質免疫沉澱配合高通量定序(Chromatin immunoprecipitation sequencing;ChIP-seq))去尋找可能受調控的基因。首先,我們挑選出14個有潛力的基因。接著,我們挑選能穩定靜默YWHAZ蛋白表現之細胞株,並進行細胞移動(migration)與侵襲(invasion)能力測試,發現YWHAZ的抑制會降低肺癌細胞移動與侵襲能力。再利用此細胞株進行即時定量聚合酶連鎖反應(QPCR)並與微陣列分析(microarray)結果比對後,確認有9個可能受YWHAZ調控之基因。而且在靜默YWHAZ表現之細胞株中再次表現YWHAZ後,可能受調控之基因表現有回復(recover)之情形。此外,進行染色質免疫沉澱配合即時定量聚合酶連鎖反應(ChIP-QPCR)實驗發現,ATF3、DMTF1、DUSP1和PAK2確實與YWHAZ-β-catenin-TCF-4蛋白複合體有交互作用;電泳移動分析結果顯示,ATF3、DMTF1、DUSP1和PAK2基因的啟動子上確實有TCF-4的結合序列。綜合以上結果,YWHAZ蛋白會經由與β-catenin和TCF-4蛋白形成的複合體來調控下游基因之表現。
URI: http://hdl.handle.net/11455/92244
Rights: 不同意授權瀏覽/列印電子全文服務
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