透過功能喪失掃瞄篩選參與膀胱癌表皮-間葉轉型過渡期之基因

Identification of the genes involved in epithelial-to-mesenchymal transition of bladder cancer by loss of function screening

胡家瑜(Chia-Yu Hu)

指導教授 ： 俞松良

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01180

摘要

膀胱癌為生殖泌尿道第二常見的惡性腫瘤，在台灣每年有超過一千例新診斷病人。超過 90％的膀胱癌被歸類為尿路上皮癌。初診個案中，約 70％∼80％為非肌肉侵入性疾病；儘管在內視鏡切除手術及膀胱灌注治療後，仍有50％∼70％的病人會復發，10％∼30％的病人會進展為肌肉侵入性疾病。截至目前為止，轉移性膀胱癌患者的預後仍然很差，平均存活時間約 12 - 15個月。上皮間質轉型過渡（EMT）在癌症的惡性發展和轉移中有著至關重要的作用；E - cadherin的表現量降低或不表現被認為是乳頭狀瘤的進展為侵襲性癌症的一個重要指標。為了找出在膀胱癌中，調控上皮間質轉型過渡的新未知基因，在此採用了可標靶幾乎所有的人類基因的82,000個shRNA匯集文庫進行了全基因組的RNA干擾掃瞄分析，期望可以挑選出增加E - cadherin啟動子活性的基因。首先，在具有顯著間質人類膀胱癌細胞株中轉染標記綠色螢光報道基因的E - cadherin啟動子，並且得到穩定轉染細胞株。之後將該穩定轉染細胞株感染慢病毒shRNA匯集文庫。病毒感染後24小時，以嘌呤黴素（puromycin）篩選有shRNA嵌入的細胞。接著，利用細胞分選儀二次分離前10％螢光表現上升的細胞族群。該分離之細胞群之後被放大培養、抽取其基因組去氧核糖核酸，再以客制化晶片進行微陣列分析。藉此，找到40個具有促進上皮間質轉型現象的潛力目標基因。

關鍵字

上皮間質轉型 ； 膀胱癌 ； 全基因組的RNA干擾掃瞄分析

並列摘要

Bladder cancer is the second most common malignancy of the genitourinary tract in the world and over 1,000 new cases in Taiwan per year. More than 90% of bladder cancer is classified as urothelial carcinoma (UC). Of newly diagnosed cases, approximately 70%~80% will present with nonmuscle‐invasive disease, and despite endoscopic and intravesical treatments, 50%~70% will recur and 10%~30% will progress to muscle‐invasive disease. The prognosis for patients with metastatic bladder cancer remains poor, with a median survival time of approximately 12~15 months. The epithelial to mesenchymal transition (EMT) plays crucial roles in cancer progression and metastasis. The loss of E‐cadherin expression is considered a hallmark in the progression of papilloma to invasive carcinoma. To identify the novel genes regulating EMT program of bladder cancer, we performed a genome‐wide RNAi screen using 82,000 pooled lentiviral shRNAs targeting almost all human genes and selected for transduced cells with increased E‐cadherin promoter activity. The human bladder cancer cell line with significant mesenchymal phenotype was first transfected with E‐cadherin promoter‐GFP reporter to generate the stable transfectants. The resulting transfectants were then infected with the pooled lentiviral shRNA library. After viral infection for 24 hours, the cells were treated with puromycin for selection of shRNA‐integrated stable cells. The top 10% in entire fluorescence‐increasing cell population was separated sequencially by cell sorter. Enriching the sorted cells by subculture for few days, the genomic DNAs were extracted from the cells with higher EGFP and further analyzed by microarray. Finally, A set of 40 genes identified to be the potential EMT regulators.

並列關鍵字

Epithelial to Mesenchymal Transition ； Bladder Cancer ； Genome-wide RNAi Screening

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