口腔癌是最常見的頭頸癌之一，有90%的口腔癌源於口腔鱗狀細胞癌。口腔癌細胞的轉移會明顯地降低口腔癌病人的存活率。在實驗室先前的研究中指出低表現量的EZH2會伴隨口腔癌細胞的高度侵襲性。EZH2是polycomb repressive complex 2的一部分並且能作為組蛋白甲基轉移酶使histone H3 lysine 27甲基化。在低侵襲性的口腔癌細胞OC3中knockdown EZH2會促進口腔癌細胞的遷移與侵襲性。我們發現EZH2可以在口腔癌細胞中作為抑癌基因，低表現量的EZH2還會造成口腔癌細胞的高遷移侵襲性。在本研究中，miRanda、Diana-microT和miRWork等線上程式被用來在miRNA array資料中過濾並選出可能會在高度侵襲性口腔癌細胞中標靶EZH2的miRNA。我們發現在高度侵襲性的口腔癌細胞中，高表現的miR-200a-3p伴隨低表現的EZH2。過量表現miR-200a-3p會降低EZH2的表現量並同時促進口腔癌細胞的侵襲性。從ChIP的實驗中，我們發現miR-200a基因在轉錄啟始點附近的H3K27me3修飾在高侵襲性的口腔癌細胞中是降低的。從ENCODE和JASPAR CORE網站資料的分析當中，我們也發現了USF2、ZBTB7A和STAT1在miR-200a轉錄起始點的結合位。ChIP的實驗中則顯示ZBTB7A會對miR-200a的轉錄進行調控。其次，IL-8的添加也會促進miR-200a-3p的表現。因此，我們發現在口腔癌細胞侵襲過程中，miR-200a-3p表現量的提昇會降低EZH2的表現量。

Oral cancer is the most common head and neck cancers and more than 90% of oral cancer results from oral squamous cell carcinoma (OSCC). OSCC cells metastasis significantly reduces survival rate of oral cancer patients. In our previous study, we found that low expressions of EZH2 associate with increased invasiveness of OSCC cell lines. EZH2, part of PRC2 complex (Polycomb Repressive Complex 2), is a histone lysine methyltransferase that adds methyl groups to the H3K27. Knockdown of EZH2 in low invasive cell line, OC3, enhances migration/invasion of OSCC cells. Accordingly, we found EZH2 can act as a tumor suppressor gene, and the reduced expression of EZH2 led to increased cell migration/invasion in OSCC cell lines. In this study, miRanda, Diana-microT and miRWork prediction software were used to screen and select miRNAs targeting EZH2 mRNA from miRNA array data. Our results suggested that high expression of miR-200a-3p correlated to low expression of EZH2 and highly invasive OSCC cell lines, and overexpression of miR-200a-3p might promote migration and invasion in oral cancer cells. By doing Chromatin immunoprecipitation (ChIP) analysis, reduced H3K27me3 marks at miR-200a promoter region and increased ZBTB7A occupancy were found to orchestrate the upregulation of miR-200a-3p in the highly invasive OSCC cells compared to OC3 cells. In addition, IL-8 was upregulated in the highly invasive OSCC cells. IL-8 treatment also upregulated the expression of miR-200a-3p. In conclusion, upregulation of miR-200a-3p might suppress EZH2 expression during oral cancer invasion.

Index
Abstract…………………………………………………………………...……………….i
中文摘要………………………………………………………………..….………….…..ii
致謝……………………………………………………………………………………….iii
Index……………………………………..…………………………….………..………..v
Abbreviation……………………………..…………………………………..………..viii
Introduction……………………………..…………………………….………..……...…1
Oral cancer…………………………………………………….……………….……1
Cancer metastasis……………………..…………………………….……….....……2
Histone modifications………………………..……………………………..……….3
Enhancer of zeste homolog 2 (EZH2) ………………………..….………..…………3
MicroRNAs (miRNAs) …………………………………………….…………….…5
MiRNAs in cancer………………………..…………………………………………5
MiR-200 family………………………..……………………………………………6
Signal transducer and activator of transcription (STAT1) ………………….………7
Zinc finger and BTB domain containing 7A (ZBTB7A) ……………………… ….8
Interleukin-8 (IL-8) ………………………..……………………………………….8
Interleukin-6 (IL-6) ………………………..……………………………………….9
Materials and Methods………………………..………………………………………..11
Vectors and reagents………………………..………………………………………11
Cell lines and cell culture………………………..…………………………………11
MiRNA, miRNA sponge construct and miRNA mimic……………………………12
Lentivirus infection and transient transfections……………………………………13
Boyden chamber assays………………………..………………………………..…13
Total RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), polymerase chain reaction (PCR), semi-quantitative real-time polymerase chain reaction (QPCR) …………………………………..………………………….……14
MiRNA quantification………………………..……………………………………14
Protein extraction and Western blot analysis…………………………………15
Chromatin immunoprecipitation (ChIP) analysis…………………………………15
MiRNAs analysis………………………..…………………………………………16
Statistical analysis………………………………………………………..………16
Results………………………..………………………………………………………18
The expressions of EZH2 are reduced in highly invasive OSCC cell lines…………18
Selection of miRNAs that may target EZH2 mRNA…………………………18
Correlation among miRNAs, EZH2 and invasion of OSCC cell lines…………19
MiR-200a-3p regulates cancer cells migration, invasion and the expression of
EZH2 in OSCC cell lines. …………………………………………………………20
H3K27me3 histone marks were reduced at miR-200a promoter…………………20
Candidate transcription factors that might regulate expression of miR-200a-3p in
the highly invasive OSCC cells………………………....…………………………21
Cytokines induced the expression of miR-200a-3p in oral cancer cells…………22
Discussion……………………………………………………………………………….24
Figures…………………………………………………………………….……………28
Figure 1. The expressions of EZH2 reduce in highly invasive OSCC cell lines…28
Figure 2. The expressions of miRNAs in OC3-IV2 compared to OC3……………29
Figure 3. Selection of miRNAs that may target EZH2 mRNA……………………30
Figure 4. MiR-138-5p is generated from miR-138-2 in OC3 cell line……….…31
Figure 5. Correlation among miRNAs, EZH2 and invasion of OSCC cell lines……32
Figure 6. Overexpression of miR-200a-3p in OC3 cell lines reduces the expression
of EZH2……………………………………………………………………………33
Figure 7. Effect of miR-200a-3p in OSCC cell lines on cell migration, invasion and
the expressions of EZH2……………………...…….………………………………34
Figure 8. H3K27me3 histone marks are reduced at the proximity of miR-200a promoter region…………………...……………………………………………..…35
Figure 9. Candidate transcription factors that may regulate expression of
miR-200a-3p in the highly invasive OSCC cells……………………...……...……37
Figure 10. ChIP analysis for candidate transcription factors………….……...……38
Figure 11. Cytokines induce the expression of miR-200a-3p in oral cancer cells…40
Figure 12. The expressions of miR-138-5p and EZH2 in OSCC cell lines………..41
Figure 13. The expressions of miR-138-5p and miR-200a-3p in OC3, C9, SCC
and SAS cell lines……………………………………………………………….…42
Figure 14. Effect of overexpressing miR-200a-3p on the expressions of
E-cadherin in OSCC cell lines…………………..…………………………………43
Figure 15. The expressions of candidate tumor promoting genes and tumor
suppressor genes in OSCC cell lines………………………………………………44
Figure 16. The expressions of EMT markers in OSCC cell lines…………………45
Figure 17. Working model for the post-transcriptional regulation of EZH2 by
miR-200a-3p…………………………………………………………….…………46
Table……………………………………………………………………………………47
Table 1. Primers sequences for miRNA RT-PCR…………………………………47
Table 2. Primers sequences for PCR and QPCR…………………………………48
Table 3. Primers sequences for ChIP analysis…………………………….………49
References………………………………………………………………………..……50

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