長片段非編碼核糖核酸 (Long non-coding RNAs) 是長度約大於200個鹼基，不會轉譯出蛋白質的核糖核酸，但會參與人類癌症細胞上，可能參與細胞的生長、凋亡、轉移之功能。目前長片段非編碼核糖核酸在口腔癌的角色與機制仍未知。在本篇研究中，我們使用次世代定序法分析人類口腔鱗狀細胞癌 (Oral Squamous Cell Carcinoma) 組織和相應癌旁正常組織的轉錄體表達譜。根據次世代定序結果我們發現，有19個長片段非編碼核糖核酸在口腔鱗狀細胞癌中表現量上升(變化倍數大於2)，20個長片段非編碼核糖核酸則表現下降(變化倍數小於-2)，由於本實驗室主要專注在甲基化調控的基因，因此高度甲基化導致表現量下降的長片段非編碼核糖核酸將成為本研究的主軸，所以我們進一步分析20個低表現的長片段非編碼核糖核酸在口腔鱗狀細胞癌中的表現量，經即時聚合酶連鎖反應定量(qPCR)顯示ATP13A4-AS1、IL20RB-AS1、LINC00265、LINC00478、LINC00568、MAST4-AS1、MIR600HG以及SOX21-AS1皆在口腔鱗狀細胞癌組織相較於癌旁正常組織之表現明顯下降。進一步評估在臨床特徵上的意義顯示，LINC00265低表現與病理晚期(P value=0.013)以及較大尺寸的腫瘤有關(P value=0.006)，其餘的並未顯示與臨床特徵有任何關聯性，而有趣的是存活曲線以及COX迴歸分析發現，SOX21-AS1 (SOX21第一反義核糖核酸) 低表現與較短的存活時間顯著相關(log rank p value = 0.016) (校正前死亡風險 0.19倍, 95%CI 0.04-0.86, P = 0.031)，進一步多變向分析將細胞分化和病理分期校正後，仍具有統計上的差異(校正後死亡風險0.19倍, 95%CI 0.04-0.87, p=0.032)。而在這兩個與臨床有關連性的長片段非編碼核糖核酸中僅有SOX21-AS1在細胞株中會因去甲基藥劑處理而回復其表現量，同時我們也發現在臨床口腔鱗狀細胞癌檢體中，SOX21-AS1上游的DNA被偵測到甲基化的比例非常高(CpG1: 85位當中有65位, 76.5%; CpG2: 85位當中有52位, 61.2%; CpG3: 85位當中有49位, 57.6%)，並且也發現SOX21-AS1甲基化在口腔鱗狀細胞癌中呈現較高的甲基化程度以及較差的細胞分化，這些數據顯示，高度甲基化可能導致SOX21-AS1在口腔鱗狀細胞癌中異常低表現，因此我們利用體外甲基化分析(in vitro methylation assay)進一步證實，SOX21-AS1的表現的確會因啟動子被甲基化而抑制，我們也利用生物資訊方式，總共有12個轉錄因子（AP2alpha, CEBPA, CEBPB, GATA-1, NF-1, NF-kB, NR3C1, RelA, SP1, TEAD2, WT1, YY1）可能會調控SOX21-AS1的轉錄活性，而這些轉錄因子與啟動子甲基化狀態之間的關係仍需要進一步實驗確認，未來我們也將進一步確認SOX21-AS1在口腔鱗狀細胞癌中的生物功能，總而言之，利用次世代定序法，我們找到許多異常的長片段非編碼核糖核酸，其中高度甲基化導致異常低表現的SOX21-AS1可作為口腔鱗狀細胞癌預後之生物標誌。

Long non-coding RNAs (LncRNAs) are more than 200 nucleotides in length; however, they lack protein transcription ability. The biological function of lncRNAs could regulate cell growth, apoptosis, and metastasis in human cancer. However, the mechanism and biological function of lncRNAs remain unknown in oral squamous cell carcinoma (OSCC). In this study, we performed the transcriptome profiles of human OSCC tissues and corresponding adjacent normal tissues from two patients by Next Generation Sequencing (NGS) approach. According to our data, we identified 19 lncRNAs were upregulated (fold change > 2) and 20 lncRNAs downregulated (fold change < -2) in OSCC compared to corresponding adjacent normal tissues. Because our laboratory is interested in DNA methylation associated genes, downregulated lncRNAs are priority to further study. Therefore, we used the quantitative real-time polymerase chain reaction (RT-PCR) to examine the expression levels of lncRNA candidates, revealing that the expression levels of ATP13A4-AS1, IL20RB-AS1, LINC00265, LINC00478, LINC00568, MAST4-AS1, MIR600HG and SOX21-AS1 significantly decreased in OSCC tissues. We further assessed the impact of these lncRNAs on clinical outcomes, showing that low expression levels of LINC00265 well correlated with late pathologic stage (P value=0.013) and large tumor size (P value=0.006). Interestingly, Kaplan-Meier curves revealed that low expression level of SOX21-AS1 (SOX21 antisense RNA 1) was significantly associated with a shorter survival (log rank p value= 0.016) (crude hazard ratio 0.19, 95%CI 0.04-0.86, p=0.031). Multivariate analysis revealed that low expression levels of SOX21-AS1 significantly associated with a shorter survival (adjusted hazard ratio 0.19, 95%CI 0.04-0.87, p=0.032). In addition, the expression levels of SOX21-AS1 could be restored in oral cancer cells with 5-aza-2'-deoxycytidine (5-aza-dc) treatment. We further analyzed the methylation status of three individual CpG islands of SOX-21-AS1 using combined bisulfite restriction assay (COBRA) and revealed that hypermethylated promoter regions of SOX-21-AS1 were frequently observed in OSCC (CpG1: 65 out of 85, 76.5%; CpG2: 52 out of 85, 61.2%; CpG3: 49 out of 85; 57.6%). Hypermethylation of SOX21-AS1 promoter was significantly correlation with moderate or poor cell differentiation of OSCC. In vitro methylation assay showed that SOX21-AS1 transactivation activity could be significantly repressed with a hypermethylated promoter. Taking together, these results indicated that abnormal DNA hypermethylation might result in SOX21-AS1 repression in OSCC. We also identified 12 putative transcription factors (AP2alpha, CEBPA, CEBPB, GATA-1, NF-1, NF-kB, NR3C1, RelA, SP1, TEAD2, WT1, YY1) using bioinformatics approach. However, the detailed roles of them still need more experiments to confirm in the future. Concluding, our study revealed that DNA hypermethylation may result in silencing SOX21-AS1 expression in OSCC. Low SOX21-AS1 expression could be provided as a good independent prognostic biomarker for OSCC.

論文審定書+i
誌謝+ii
Abbreviations+iii-iv
摘要+v-vi
Abstract+vii-ix
Contents+x-xii
Introduction+1
1. Oral cancer+1
2. LncRNAs mechanism+1-3
3. LncRNAs in oral cancer+3-5
4. LncRNA, methylation and cancers+5-6
5. Next generation sequencing (NGS) and cancer+6-7
6. Specific aims+8
Materials and methods+9
1. Patients and tissue samples+9
2. Cell lines+9-10
3. Section, staining and laser capture microdissection (LCM) +10
4. Hematoxylin and eosin stain+10
5. Extraction of RNA from LCM cells for NGS+10-11
6. NGS+11
7. NGS analysis+11-12
8. DNA and RNA extraction from patient tissues+12
9. 5-aza-dC and TSA treatment+12-13
10. RT-PCR of lncRNAs+13
11. DNA bisulfite conversion+13
12. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis+14
13. SOX21-AS1 promoter construction+14-15
14. In vitro methylation+15
15. Luciferase assay+15
16. Nuclear/cytoplasmic RNA fractionation+16
17. Statistical analysis+16-17
Results+18
1. LncRNA profiling of oral squamous cell carcinoma+18-19
2. Expression of lncRNA candidates identified from NGS in OSCC patients+19
3. The correlation between lncRNA expression and clinicopathologic characteristics+19-20
4. DNA Silencing SOX21-AS1’s promoter activity+20-23
5. Predicted transcription factors binding to the SOX21-AS1 promoter+23-24
6. Cell RNA fractionation for confirming location of SOX21-AS1+24
7. Summary+24-25
Discussion+26-33
References+34-42
Table+43-55
Figures+56-71

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