上皮細胞中角質蛋白會形成在中間絲以調節細胞形狀、流動性、膜運送和訊息傳遞。雖然角質蛋白6（K6）和角質蛋白14（K14）在某些鱗狀細胞癌中高表達，並已被建議作為腫瘤標誌物，但角質蛋白如何影響癌症發展的分子機制楚我們仍不清楚。在本研究中，在口腔鱗狀上皮細胞癌(Oral Squamous Cell Carcinoma,OSCCs)中我們利用了次世代定序以及螢光原位雜交法證明了角蛋白6與角蛋白14嵌合體的存在。並確定出兩種獨特的融合類型（類型1和類型2），共有21種不同的基因融合變體在OSCCs被驗證。並且在臨床腫瘤檢體中檢測K6-K14基因融合的高比率：第一型的25％與第二型的33％。探討角質蛋白基因融合的原因，我們利用口腔鱗狀上皮細胞癌的致癌物質的組合檳榔鹼、尼古丁以及酒精處理。我們的數據中發現檳榔鹼與尼古丁會加速誘導K6-K14基因融合。功能研究進一步證實了角蛋白的第二型融合可以促進上皮細胞向間充質細胞轉化的現象和癌症幹細胞的形成以及增加細胞的增殖、遷移和侵襲的能力。因此，我們的研究發現角蛋白基因融合涉及致癌的新機制。

Keratin cytoskeleton proteins form intermediate filaments in epithelial cells to regulate cell shape, mobility, membrane trafficking and cellular signaling. Although keratin-6 (K6) and -14 (K14) are highly expressed in certain squamous cell carcinomas and have been suggested as tumor markers, molecular mechanisms of how keratins contribute to cancer development still remain elusive. In early studies, we demonstrated novel K6-K14 chimeras in oral squamous cell carcinomas (OSCCs) by pair-ended transcriptome sequencing and subsequent validation by fluorescence in situ hybridization and junction site mapping. Two unique fusion types (type-1 and type-2) were identified with a total of 23 in-frame fusion variants verified in OSCCs. Clinical screening confirmed high detection rate of K6-K14 fusions in tumor samples: 33% for type-1 and 25% for type-2. To know the cause for the keratin fusions, OSCC cells were treated with arecoline, 4NQO, alcohol, and the combinations of these three well-known OSCC carcinogens. Our data found acrecoline as the promoter to accelerate the induction of K6-K14 fusions by 4NQO. Functional study further confirmed that the type-2 fusions can increase cell proliferation, migration and invasion via EMT and cancer stem cell formation. Our study thus uncovered a novel mechanism involved in carcinogenesis by keratin fusions.

論文審定書------------------------------------------------------------------------------------------i
誌謝 (Ackowledgements) ------------------------------------------------------------------------ii
中文摘要 (Chinese of abstract) ----------------------------------------------------------------iii
英文摘要 (English of abstract) -----------------------------------------------------------------iv
縮寫表(Abbreviations) ----------------------------------------------------------------------------v
前言 (Introduction) -------------------------------------------------------------------------------1
材料與方法 (Materials and Methods) ---------------------------------------------------------5
實驗結果 (Results)--------------------------------------------------------------------------------8
討論 (Discussion)--------------------------------------------------------------------------------14
圖表 (Figures)------------------------------------------------------------------------------------19
圖1、識別染色體易位t(12; 17)(q13;q21)在口腔鱗狀上皮細胞癌(OSCC) --------20
圖2、將基因融合KRT6-KRT14轉錄在口腔鱗狀細胞癌進行鑑定-----------------21
圖3、KRT6-KRT14基因融合在口腔鱗狀上皮細胞癌產生變異---------------------22
圖4、qPCR檢測KRT6-KRT14基因融合變異在鱗狀上皮細胞癌------------------23
圖5、臨床資料中顯示KRT6-KRT14基因融合變異與口腔鱗狀細胞癌之相關--24
圖6、K6-K14基因融合V9型態影響癌細胞增生--------------------------------------25
圖7、K6-K14基因融合V9型態促進下游訊號路徑及EMT現象------------------26
圖8、K6-K14基因融合V9型態促進EMT現象---------------------------------------27
圖9、K6-K14基因融合V9型態透過EMT去影響細胞的移動---------------------28
圖10、K6-K14基因融合V9型態透過EMT去影響細胞的侵襲-------------------29
圖11、K6-K14基因融合V9型態促進細胞產生抗Cisplatin的能力---------------30
圖12、KRT6-KRT14基因V9融合型態增強致腫瘤性--------------------------------32
圖13、3D培養環境下，KRT6-KRT14基因V9融合型態增強致腫瘤性----------33
圖14、KRT6-KRT14基因V9融合型態會增進癌幹細胞的表現--------------------34
圖15、3D培養環境下，KRT6-KRT14基因V9融合型態促進癌幹細胞的表現--35
參考資料(References)--------------------------------------------------------------------------- 36

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