口腔癌為頭頸癌的一種,為台灣第六大癌症,百分之九十以上的口腔癌屬於口腔鱗狀 上皮細胞癌。在歐美國家,口腔癌的肇因主要為長期抽煙、嚼食煙草、喝酒等行為。而在 台灣與東南亞國家,口腔癌的發生則與嚼食檳榔有高度的關聯性。因此,了解嚼食檳榔所 導致的口腔癌致病機轉,對於台灣的口腔癌治療是相當重要的。表觀遺傳學為不經由改變 核酸序列,而使基因表現發生變化的調控機制,許多癌症的發生與異常的表觀遺傳調控有 高度的關聯性。PRC2 複合物在基因的調控上扮演重要角色,其成員 EZH2 為 H3K27 的三 甲基轉移酶,常見過量表現於多種癌症以抑制抑癌基因表現。然而,EZH2 在口腔癌中的 異常表現及其機制仍不明確,此論文研究目的為探討 EZH2 在口腔鱗狀上皮細胞癌中的角 色。比較各種口腔癌細胞株, EZH2 的表現量在 OC3 細胞株有較高的情形。為了探討增 加的 EZH2 在 OC3 細胞株中所扮演的調控角色,本研究利用 shRNA 抑制 OC3 細胞株的 EZH2 表現。研究結果顯示,抑制 EZH2 表現的 OC3 細胞株相較於控制組有較強的侵襲與 遷移能力。另外,本研究進一步發現,抑制 OC3 細胞株的 EZH2 會使 ADAMTS1 與 MMP3 的 RNA 表現量增加。總結此篇論文研究結果,有別於其他癌症,EZH2 在 OC3 口腔癌細 胞株中很可能透過抑制 ADAMTS1 與 MMP3 的表現,以抑制癌細胞侵襲與遷移的能力。

Oral cancer, the sixth most common cancer in Taiwan, is the major subtype of head and neck cancer, and more than 90% of oral cancer are oral squamous cell carcinoma (OSCC). Smoking, tobacco chewing and alcohol consumption are the major risk factor of OSCC in America and European countries. In Taiwan and most of the Southeast countries, betel nut chewing is highly associated with OSCC. Understanding the mechanisms of betel nut chewing-related OSCC is significant for oral cancer treatment in Taiwan. Aberrant epigenetics regulation is one of the hallmarks of cancer. Epigenetics modification regulates gene expression without the change of nucleotide sequence. Polycomb repressive complex 2 (PRC2) is a key epigenetic regulator required for gene silencing during development and cancer. Recent studies suggest that EZH2, the histone methyltransferase of PRC2, represses tumor suppressor genes by mediating trimethylation of histone H3 at lysine 27 (H3K27me3) in variety of cancer, including head and neck cancers. However, the anomaly of EZH2 expression and its underlying mechanisms in OSCC is still unclear. The aim of the present study is to investigate the role of EZH2 in OSCC. Comparing various OSCC cell lines, EZH2 was found elevated in human oral carcinoma 3 (OC3) cells. To examine the significance of the increased EZH2 in OC3 cells, we established OC3-shLacZ and OC3-shEZH2 stable cell lines. Intriguingly, the migration and invasion ability of OC3-shEZH2 cells increased compared to control OC3-shLacZ cells. Our findings suggest that EZH2 may suppress migration and invasion ability of oral cancer by inhibiting matrix metalloproteinase-3 (MMP3) and a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) expression.

Abstract I
中文摘要 II
致謝 III
Index V
Abbreviations VIII
Introduction 1
Materials and Methods 11
Antibodies and reagents 11
Cell lines and cell culture 12
Knockdown of EZH2 using lentivirus expressing shRNA constructs 12
MTT assays 13
Migration and invasion assays 13
Total RNA extraction, reverse transcription polymerase chain reaction (RT-PCR) and semi-quantitative real-time polymerase chain reaction (Q-PCR) 14
Protein extraction and western blotting 14
Chromatin immunoprecipitation (ChIP) analyses 15
Statistical analysis 16
Results 17
The protein expression level of EZH2 is higher in OC3 cells compared to other oral cancer cell lines 17
The mRNA expression level of EZH2 is higher in oral squamous cell carcinoma compared to normal tissue 17
Knockdown of EZH2 does not affect cell proliferation of OC3 cells 18
Knockdown of EZH2 enhances migration of OC3 cells 19
Knockdown of EZH2 promotes invasion of OC3 cells 20
EZH2 mRNA level of metastatic OSCC is lower than the non-metastatic OSCC 21
Knockdown of EZH2 does not alter the mRNA expression of TIMP family, E-cadherin, SLIT2, DAB2IP and RAP1GAP 21
Expression levels of MMP3 and ADAMTS1 in EZH2-knockdown OC3 cells 22
The increased ADAMTS1 expression in OC3-shEZH2 cells is correlated with H3K27me3 modification of ADAMTS1 promoter 23
Discussion 25
Figures 29
Figure 1. The EZH2 expressions in oral squamous cell carcinoma (OSCC) cell lines 29
Figure 2. EZH2 mRNA expressions in HNSCC and OSCC patients 31
Figure 3. Knockdown of EZH2 expression by shRNA in OC3 cells 32
Figure 4. Morphology of OC3-shLacZ and OC3-shEZH2 cells 34
Figure 5. Knockdown of EZH2 does not affect the proliferation rate of OC3 cells 35
Figure 6. Migration of OC3-shLacZ and OC3-shEZH2 cells 36
Figure 7. Invasion of OC3-shLacZ and OC3-shEZH2 cells 37
Figure 8. EZH2 mRNA levels of OSCC patients with or without lymph node metastasis 38
Figure 9. Expression levels of TIMP family, E-cadherin, SLIT2, DAB2IP and RAP1GAP in OC3-shLacZ and OC3-shEZH2 cells 40
Figure 10. Expression level of NF-κB, TNF, IL-6, IL-8, WNT5A, WNT5B, ADAM17, MMP3 and ADAMTS1 in OC3-shLacZ and OC3-shEZH2 cells 42
Figure 11. ChIP analysis of H3K27me3 on ADAMTS1 and MMP3 promoter 43
Table 44
Table 1. Primer sequences for PCR 44
Table 2. Primer sequences for Q-PCR 46
Appendix 47
Fig A1. mRNA level of EZH2 in metastatic breast and prostatic cancer 47
Reference 48

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