臺灣博碩士論文加值系統

目前在台灣口腔鱗狀上皮癌(oral squamous cell carcinoma，OSCC)的致死率排名已躍身為第四名；正常的口腔細胞變成癌前病灶再惡性轉型成口腔癌的過程中是需要經過許多的刺激和調控。而其中為口腔癌前病變之一的疣狀增生(verrucous hyperplasia，VH) 具有高度惡性轉形的能力。因此，發展可用來預測疾病的分子標記對於改善和診斷疾病是必要的。MicroRNAs (miRNAs) 為小片段非編碼的RNA，其長度約為22個核苷酸所構成，在癌症中可扮演抑癌基因或是致癌基因的角色。因此，我們的研究目的想要探討與口腔鱗狀細胞癌相關的miRNAs在癌化過程中所扮演的分子機制為何。先前由VH與正常口腔黏膜組織miRNA microarray分析發現miR-133a在VH檢體表現量較低，我們首先利用real-time PCR分析，發現與正常的細胞和組織相比，miR-133a在口腔癌細胞株、VH以及OSCC檢體中，都有顯著的下降。我們同時也大量表現miR-133a在口腔癌細胞株，發現miR-133a對細胞生長速度並沒有顯著的影響，但對於口腔癌細胞株的爬行和侵襲能力則因不同的細胞株有不同的影響。另外，我們利用原位雜交染色(in situ hybridization，ISH) 來偵測miR-133a在臨床檢體的表現，發現miR-133a確實在OSCC表現量較低，而且同時也發現在分化的組織miR-133a的表現量比較未分化的組織來的高。利用cDNA微列陣技術及生物資訊軟體分析miR-133a可能的目標基因，我們發現有兩個可能的目標基因(AFAP1L2 和 SERPINH1)與miR-133a的表現量呈反比。在口腔癌細胞大量表現miR-133a的時候，觀察到AFAP1L2和 SERPINH1表現量會被抑制，這些目標基因的3端非轉譯區(3’-UTR) 預測皆含miR-133a鍵結區。從以上的實驗發現miR-133a在口腔癌癌化過程中可能扮演抑癌基因的角色。而更進一步想要探討在口腔癌癌化過程中miR-133a分子機制，或許可在口腔癌中可以當作一個新的治療途徑。

Oral squamous cell carcinoma (OSCC) is the fourth leading cause of male cancer death in Taiwan. OSCC development usually involves multistep progression from normal oral mucosa changing to oral precancerous lesions and then changing to OSCC. In which, oral verrucous hyperplasia (VH) is one of oral precancerous lesions with relatively high malignant transformation potential. Therefore, identification of molecular markers which can predict disease progression is necessary. MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides that can function as oncogenes or tumor suppressors. Thus, the objective of this study is to elucidate the molecular and biological roles of OSCC-associated miRNAs for a better understanding in oral cancer progression. We found that the expression level of miR-133a was reduced in oral cancer cells, clinical VH and OSCC specimens by a qPCR analysis. Overexpression of miR-133a in OSCC cell lines has no effect on cell proliferation but while having differential expression on migration and invasion depending on the cell types. The expression pattern of miR-133a in clinical OSCC specimens was further examined by an in situ hybridization (ISH). Interestingly, we found that the expression of miR-133a was high in differentiated oral cancer cells and normal tissues. In addition, we also analyzed cDNA microarray and used bioinformatics to predict targeted genes of miR-133a, and found two oncogenes (AFAP1L2 and SERPINH1) might be negatively regulated by miR-133a. MiR-133a transfection reduced expression level of mRNA of these genes, and suggested these genes have potential binding sites of miR-133a. These findings suggested that miR-133a could influence cell motility by targeting potential oncogenes in oral cancer. Further study to explore the molecular mechanism of miR-133a in oral cancer progression may provide beneficial effects on oral cancer chemotherapeutics and prevention.

摘要 I
Abstract II
Acknowledgements III
Contents V
Abbreviation VIII
Introduction 1
1. Oral cancer 1
2. MicroRNA 2
3. MicroRNA and cancer 3
MicroRNAs as Tumor Suppressors 3
MicroRNAs as Oncogenes 4
Therapeutic development of microRNAs 4
4. MiR-133a 5
Predicting candidate genes for miR133a 5
Rational and Specific aims 7
Materials and methods 8
Clinical samples and cell culture 8
In situ hybridization (ISH) 9
MicroRNA Quantitative real-time PCR 10
Transient transfection 10
Cell proliferation (WST-1 assay) 11
Cell migration and invasion assay 11
Wound healing assay 12
Prediction of microRNA targets 12
Quantitative real-time PCR analysis 13
Western blot analysis 13
Statistical analysis 14
Results 15
1. Expression of miR-133a was down-regulated in oral cancer compared to normal tissues. 15
2. MiR-133a was highly expression in normal tissue and associated with cancer cell differentiation. 15
3. MiR-133a does not influence the proliferation of OSCC cells but significantly influences cancer cell motility. 16
4. AFAP1L2 and SERPINH1 are potential target genes of miR-133a in OSCC cell lines. 17
Discussions 18
Conclusions 22
References 23
Table 28
Figure 30
Figure 1. Expression profile of miRNAs in oral cancer tissues. 30
Figure 2. Expression of miR-133a in clinical specimens and oral cancer cell lines. 31
Figure 3. Expression of miR-133a in clinical specimens was examined by in situ hybridization. 33
Figure 4. 34
Figure 5. Overexpression of miR-133a in oral cancer cell lines has no effect on cell growth. 36
Figure 6. Overexpression of miR-133a in oral cancer cell lines influences cancer cell migration and invasion. 37
Figure 7. Overexpression of miR-133a in oral cancer cell lines enhanced wound-induced cell migration. 38
Figure 8. AFAP1L2 and SERPINH1 are potential target genes of miR-133a. 39
Appendix 40
Curriculum vitae 43

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