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研究生:吳怡璇
研究生(外文):Yi-Hsuan Wu
論文名稱:微型核醣核酸31*在口腔鱗狀上皮癌細胞中的角色及其分子機制之探討
論文名稱(外文):The role and the molecular mechanism of miR-31* in oral squamous cell carcinoma cells
指導教授:林姝君林姝君引用關係
指導教授(外文):Shu-Chun Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:137
中文關鍵詞:口腔癌微型 RNA
外文關鍵詞:oral cancerOSCCmiRNAmiR-31*RhoAcyclin B1
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在台灣,口腔癌是男性十大癌症的第四位,根據衛生署的統計資料顯示,從民國八十一年起口腔癌的發生率及死亡率逐年提升;因此,了解口腔癌的致癌機轉與進程是降低口腔癌惡化的一個重要方向。
許多癌症研究的文章指出,微小RNA (microRNA) 具有調控細胞內致癌基因及抑癌基因的表現,與癌症生成具高度相關性(1)。microRNA是一種長約 20 ~ 22 個鹼基對的 RNA,可與目標基因 (target gene) 3端非轉錄區 (3’untranslated region, 3‘UTR) 結合,進而抑制目標基因的轉錄作用。過去對microRNA的研究中,顯示 microRNA 在成熟的過程中只有引導股 (guide strand)(miR) 會與目標基因結合而抑制轉錄的進行,另一股過客股 (passenger strand)(miR*) 則會被降解而不具功能。但 2009 年 Jazdzewski 等人發表的文章中指出,microRNA的過客股也會在細胞內表現並具有其功能(2),顯示出microRNA的過客股亦具有調控基因表現的功能。
本實驗室先前的研究已證實在口腔癌細胞株 SAS 內大量表現 miR-31 前驅物 (precursor),具有促進細胞增生 (proliferation) 、爬行 (migration) 以及不貼附生長 (anchorage-independent growth) 等惡性轉表徵。由於 miR-31 前驅物在進一步的剪接修飾後會同時形成 miR-31及 miR-31*,因此本研究主要探討 miR-31 前驅物是否會透過 miR-31* 來影響細胞。研究發現,miR-31* 能夠穩定存在於口腔癌細胞內,並且過度表現之 miR-31* 具有抑制口腔癌細胞株增生、爬行之能力;進一步的研究指出,過度表現之 miR-31* 可降低細胞內 cyclin B1 以及 RhoA 蛋白質表現,故推測 miR-31* 可能透過調控 cyclin B1 以及 RhoA 基因來改變細胞之表型。由於 miR-31* 具有降低癌細胞增生與爬行之能力,未來具有發展為口腔癌治療藥物的潛力。

In Taiwan, oral cancer is the fourth leading male cancer. According to the statistics of our Department of Health, the incidence and mortality rate of oral cancer have been increased significantly since 1992. Therefore, understanding the mechanisms of tumorigenesis and progression of oral cancer becomes an important objective in dealing with this major healthy threat to our people.
Rising evidence indicates the important roles of microRNAs in tumorigenesis. MicroRNAs (miRNAs) are small non-coding RNAs, which contain 20 to 22 nucleotides. It can inhibit translation and cause degradation of target mRNAs by pairing with their 3’-UTRs (3’ untranslated region).
Substantial studies emphasized the regulatory roles of guide strands (miRs) of microRNAs. The other passenger strands (miR*s), which are generated during the processing of precursor microRNAs, are considered as non-functional by-products and will be degraded eventually. However, a recent study by Jazdzewski showed the stable expression and functions of these passenger strands. Their results implicated potential regulatory roles may exist among these passenger strands.
Our previous studies revealed overexpressed precursor of miR31 in SAS, an OSCC cell line, would enhance its malignant phenotypes, such as cell proliferation, migration, invasion, and anchorage-independent growth. Since processing of precursor of miR31 would generate not only miR-31 (guide strand) but miR-31* (passenger strand), this study is aimed primarily to investigate the roles of miR-31* in regulating the phenotypes of OSCC cells. Our present study showed miR-31* was able to stably express in OSCC cells. Moreover, over-expressed miR-31* would inhibit cell proliferation and migration, which were probably mediated by its ability to target and decrease the expression of cyclin B1 and RhoA. In summary, our results point to a potential application of the miR-31*-based target therapy for oral cancer in the future.

目錄 i

中文摘要 iv

Abstract vi

圖目錄 viii

附圖目錄 xi

附表目錄 xii

壹、緒論 1
一、口腔癌 1
二、微型RNA 4
三、微型RNA與腫瘤 11
貳、研究動機與目標 14
叁、實驗材料與方法 16
一、細胞培養 16
二、病毒組裝 (Virus particle packaging) 18
三、病毒感染 ( infection ) 20
四、RNA之萃取 ( RNA extraction) 20
五、微型RNA之反轉錄即時定量聚合酶連鎖反應 (Real-time PCR) 21
六、報導者質體的建立 (reporter plasmid construction) 23
七、報導者分析法 (Reporter assay) 29
八、微型RNA之模擬物及抑制物 (microRNA mimic and blocker) 31
九、報導者質體以及微型RNA模擬物、抑制物之轉染法 (Reporter plasmid and microRNA mimic,inhibitor transfection) 31
十、細胞增生分析法 (Cell proliferation assay) 33
十一、細胞爬行分析法 (Cell migration assay) 33
十二、細胞非貼附生長分析法 (Cell anchorage-independent growth assay) 35
十三、反轉錄聚合酶連鎖反應 (Reverse-transcription PCR) 35
十四、西方墨點法 (western blot) 37
十五、統計 41
肆、研究結果 42
一、探討 miR-31* 是否存在於口腔癌細胞株內並進一步探究其活性程度 42
二、過度表現 miR-31* 對口腔癌細胞株細胞表型影響之分析 46
三、探討在口腔癌細胞株內 miR-31* 調控的分子路徑 51
伍、討論 64
陸、圖 72
柒、參考文獻 100
捌、附圖 105
玖、附表 111

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