臺灣博碩士論文加值系統

中文摘要

口腔癌是全球盛行之癌症，研究其病程、訊息調控機轉為重要課題。微型RNA為小型之非轉譯RNA，參與各種生理及病理的調控，亦對口腔癌進程扮演重要角色。miR-125家族已被報導參與許多癌症的進程，在大腸癌及乳癌，miR-125為腫瘤抑制者；然而在非小細胞肺癌及血癌中，miR-125為腫瘤促進者，故其致病角色仍有爭議。雖然研究顯示在口腔癌患者中，miR-125的表現似較低，但miR-125在口腔癌中的功能角色及其訊息影響仍未明朗。經由軟體預測，miR-125的下游標的物可能為FAM213A (Family with sequence similarity 213, member A)。FAM213A屬於抗氧化蛋白，當細胞處於氧化壓力時，FAM213A具有保護細胞的作用。本實驗室先前研究發現，口腔癌腫瘤中具有較高的FAM213A表現。本研究顯示，不論在口腔癌細胞株中，或者病患癌組織中，均具有較低的miR-125表現，miR-125b低表現在I及II期口腔癌病患中有較差之預後有關。更證明miR-125b於口腔癌中直接標的FAM213A 3’ UTR抑制FAM213A表現，並增加細胞氧化壓力。在口腔癌細胞中過度表現miR-125b降低口腔癌細胞致癌表型，此外也確認FAM213A與miR-125b有相反之表型。本研究已建立過度表現完整FAM213A轉錄物(transcript)之細胞株，將進行FAM213A救援miR-125b及動物試驗，進一步證明miR-125b-FAM213A調控軸在口腔癌化之角色。

Abstract

Oral carcinoma (OC) is a globally prevalent cancer. To understand its disease processes and the mechanisms of signaling regulation are critical issues. MicroRNAs (miRNAs) are small non-coding RNAs, involved in the modulation of physical or pathological properties. miRNAs are also important regulators in OC pathogenesis. miR-125 family genes are involved in the disease process of a wide variety of cancers. In colorectal cancer and breast cancer, it acts as tumor suppressor. However, in non-small cell lung cancer and leukemia, miR-125 is oncogenic. Thus, there are controversies regarding the functions of miR-125 in different types of cancers. It appears that miR-125 is down-regulated in OC, but the carcinogenic mechanisms and the signaling impacts of miR-125 in OC are still obscure. In silico modules predict that FAM213A (Family with sequence similarity 213, member A) is a potential target of miR-125. FAM213A is a known anti-oxidant protein that protects cell from oxidative stress. Our previous study revealed that FAM213A was up-regulated in OC tumors. This study further specifies that miR-125 expression is down-regulated in OC tumors and cell lines. Besides, lower miR-125b expression in OC tissues is associated with the worse prognosis in stage I and II patients. We also identified that miR-125b targeted the 3’UTR of FAM213A to repress its gene activity. This targeting is accompanied with the increase of oxidative stress. Exogenous miR-125b expression decreased the oncogenicity of OC cell. miR-125b and FAM213A drive opposite oncogenic phenotypes. OC cell clones stably express complete FAM213A transcript have been established in our work. We are about to carry out rescue experiments and tumorigenic studies using these clones to further specify the roles of miR-125b-FAM213A regulatory axis in OC induction.

目錄 i
Abstract iv
中文摘要 v
壹、緒論 1
一、口腔癌 (oral squamous cell carcinoma, OSCC) 1
二、微型核糖核苷酸（micro RNA, miRNA） 1
三、微型RNA-125 (miR-125) 2
四、FAM213A (Family with sequence similarity 213,member A) 4
五、細胞活性氧化物質 (Reactive oxygen species, ROS) 5
貳、研究動機與目標 7
一、研究動機 7
二、研究目標 7
參、材料與方法 8
一、細胞培養 (Cell culture) 8
二、轉染 (Transfection) 9
三、RNA萃取 (RNA extraction) 10
四、反轉錄反應（Reverse Transcription, RT） 11
五、即時聚合酶鏈鎖反應 (Real-time PCR) 12
六、質體DNA選殖 (Gene cloning) 13
七、質體轉型 (Plasmid transformation) 16
八、少量質體DNA製備 (Mini-prep) 16
九、大量質體DNA製備 (Midi-prep) 17
十、病毒製備與感染 (Virus-prep and infection) 17
十一、報導者分析試驗 (Reporter assay) 18
十二、西方點墨法 (Western blot) 19
十三、細胞存活率分析 (MTT assay) 22
十四、細胞移行試驗 (Cell migration assay) 22
十五、細胞入侵試驗 (Cell invasion assay) 23
十六、細胞非貼附性生長試驗 (Anchorage-independent colony formation assay) 23
十七、檢測細胞活性氧化物質 (ROS detection) 24
十八、檢測蛋白氧化壓力 (Oxidized protein detection) 25
十九、口腔病理組織 (Oral tissues) 26
二十、組織晶片 (Tissue array) 26
二十一、原位雜合法 (In situ hybridization, ISH) 26
二十二、統計方法 (Statistical analysis) 28
肆、結果 29
一、口腔癌細胞株中miR-125b的表現量較正常細胞株低 29
二、口腔癌組織中有較低的miR-125b表現 29
三、過度表現miR-125b降低口腔癌細胞表現型能力 29
四、miR-125b低表現與口腔癌病患較差之預後有關 30
五、口腔癌細胞中FAM213A的表現受miR-125b所調控 31
六、miR-125b直接標的FAM213A 3’ UTR抑制FAM213A表現 33
七、FAM213A表現與口腔癌細胞表現型相關 34
八、建立FAM213A過度表現細胞株 35
九、miR-125b抑制FAM213A的表現而增加細胞氧化壓力 36
伍、討論 37
陸、圖列 43
圖一、口腔癌細胞株中miR-125a與miR-125b的mRNA表現 43
圖二、口腔癌組織中miR-125a與miR-125b的表現 44
圖三、過度表現miR-125b對SAS細胞表現型能力之影響 46
圖四、分析口腔癌miR-125b表現與病患腫瘤進程之關係 47
圖五、分析口腔癌miR-125b表現及頸部淋巴結轉移與病患存活率之關係 49
圖六、口腔癌細胞株中FAM213A的mRNA表現 50
圖七、口腔癌細胞中miR-125b與FAM213A的表現呈現負相關 51
圖八、口腔癌細胞中miR-125b過度表現抑制FAM213A表現 52
圖九、口腔癌細胞中miR-125b受到抑制時FAM213A表現量上升 53
圖十、miR-125b直接標的3’ UTR抑制FAM213A 54
圖十一、SAS細胞FAM213A基因剔除對細胞表現型能力之影響 55
圖十二、SAS細胞過度表現FAM213A對細胞表現型能力之影響 57
圖十三、建立FAM213A過度表現SAS及FaDu細胞子株 59
圖十四、miR-125b對SAS細胞內活性氧化物質之調控 60
圖十五、miR-125b及FAM213A對SAS細胞內蛋白氧化壓力之調控 61
柒、表列 62
附表一、腫瘤分期 62
附表二、Stage grouping(綜合TMN分期) 64
附表三、各項溶液配方 64
1. 10X PBS 64
2. PMSF 64
3. Lysis buffer 65
4. 4X Lower buffer 65
5. 10% Lower gel 65
6. 4X Upper buffer 65
7. 4% Upper gel 66
8. SDS sample dye 66
9. 10X Running buffer (pH 8.3) 66
10. Transfer buffer 66
11. PBST 67
12. Blocking buffer 67
13. LB培養液 67
附表四、初級抗體 67
附表五、次級抗體 67
附表六、引子序列 68
附表七、TaqMan® MicroRNA Assays所使用之探針 69
附表八、購入之FAM213A質體 69
附表九、ISH所使用之探針 69
附表十、MiRWalk預測FAM213A為miR-125b標的基因 70
捌、附圖列 71
附圖一、pBABE-puro質體 71
附圖二、pMIR-REPORTTM Luciferase質體 71
附圖三、TargetScan預測FAM213A為miR-125標的基因 72
附圖四、確認FAM213A基因剔除細胞株於口腔癌細胞株SAS中建立 72
玖、參考文獻 73

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