臺灣博碩士論文加值系統

先前研究指出，癌症的病程中往往伴隨著表觀遺傳的改變，透過核小體上標記的轉換，及microRNA的異常表現，進而影響癌症細胞的表徵。離胺酸特異性去甲基化酶 (Lysine-specific demethylase ; LSD1)是第一個被發現的組蛋白去甲基酶，其高度表現於許多腫瘤組織中，透過去甲基化活性影響某些抑癌基因 (tumor suppress genes; TSG) 的表現。但以往的研究只著重於LSD1 對於protein-coding-gene的調控。對於LSD1和microRNA在癌症的關係仍不清楚
在本研究中，我們透過系統性分析探討270個microRNA在4對膀胱癌組織的表現，發現mir-17-92 cluster高度上升於膀胱癌組織中，且抑制LSD1也同時抑制這群miRNAs於膀胱癌細胞株的表現，過去研究認為miR-17-92群簇具有促進細胞週期的功能，我們也透過功能性的分析，發現LSD1會調控細胞的生長，並影響癌細胞群落生成的現象，暗示LSD1可能透過正調節miR-17-92 群簇的表現,進而促進癌症細胞的生長。在此研究結果中顯示LSD1具有影響miR-17-92 群簇的表現，因此我們進一步的去探討LSD1如何去調控此群microRNA的機制。
先前的研究中已證實，在miR-17-92的序列上具有c-Myc 的特定結合位置，當c-Myc結合上去時會促使miR-17-92群簇的轉譯，進而調控細胞細胞的周期和生長等。由實驗結果中也顯示c-Myc亦會去影響miR-17-92群簇的表現。
除此之外，在先前的研究中發現，當c-Myc結合到特定的序列(E-Box)上時，會吸引LSD1的結合，並促使c-Myc誘導基因的表現。藉由先前研究的證實，我們推測LSD1對於miR-17-92 群簇的調控須透過c-Myc的調節，同時在研究探討中我們也觀察到在LSD1受到抑制的情況下，對於此細胞中c-Myc 基因的表現會有些微的影響，但對於c-Myc的蛋白表現則大幅的減少。藉由觀察到的現象，意味著 LSD1是否藉由調控c-Myc進而影響miR17-92群簇的表現，因此我們進一步的想去探討LSD1如何影響c-Myc蛋白的表現及調控機制。首先我們先釐清LSD1是否影響c-Myc mRNA或蛋白的降解速率，由結果顯示LSD1會影響在c-Myc蛋白層面，在缺乏LSD1的細胞，其c-Myc蛋白會透過蛋白酶體的作用加速的被分解。接著我們將去探討LSD1如何去調控c-Myc蛋白維持在細胞中的穩定性，藉由LSD1抑制劑 2-PCPA，抑制LSD1的活性後，探討相較於siRNA LSD1對於c-Myc蛋白的影響。由結果顯示，減緩膀胱癌細胞中c-Myc蛋白的降解速率，需要LSD1 蛋白的存在，但並非透過LSD1的活性去維持c-Myc蛋白的存在。
藉由以上的實驗結果，我們推論在膀胱癌細胞中LSD1會維持c-Myc蛋白的穩定性，減緩降解速率，使細胞維持一定表現量的c-Myc蛋白並促使miR-17-92 群簇的表現，進而去調控細胞的生長。

Lysine-specific histone demethylase 1 (LSD1) is the first histone demethylase discovered. The enzyme catalyzes demethylation of histone H3-lysine-4 and H3 lysine-9 and modulates the expression levels of a subset of protein-coding genes. LSD1 plays an essential role in mammalian development and is involved in many biological processes. Dysergulation of LSD1 has also been reported in several types of solid tumor, including bladder cancer. However, the effect of LSD1 on miRNA expressions and their roles in bladder cancer progression has not been explored. In our previous study, we used multiplexed stem-loop RT-PCR to profile the expression levels of 270 miRNAs in bladder cancer. We discovered that several microRNAs from the miR-17-92 cluster, a well-documented oncogenic microRNA locus, were significantly up-regulated in bladder cancer. Interestingly, depletion of LSD1in bladder cancer cells inhibited expression level of miR-17-92 cluster and cell proliferation. Previous studies have shown that c-Myc can recruit LSD1 to regulate down-stream target genes. c-Myc-binding sites are present on the promoter of miR-17-92 cluster. Therefore, we examined whether LSD1 control proliferation of bladder cancer cells through the c-Myc-regulated miR-17-92 expression. Our results showed that depletion of LSD1 or c-Myc inhibited cell growth and suppressed the expression level of miR-17-92 in bladder cancer cell lines. Depletion of LSD1 significantly reduced c-Myc protein level but had little effect on the c-Myc mRNA level. To clarify how LSD1 affects c- Myc protein level, we compared c-Myc mRNA and protein half-life in the control and LSD1-depleted cells. We demonstrated that half-life of c-Myc protein was significantly shortened in cells treated with si-LSD1. The effect of LSD1 siRNA on c-Myc protein stability was not mimicked by small molecule LSD1 inhibitor 2-PCPA, suggesting that LSD1 protein, rather than the enzymatic activity of LSD1, was required to maintain c-Myc protein stability. These results showed that elevated LSD1 could regulate growth of bladder cancer cells by stabilizing c-Myc protein and the elevated c-Myc subsequently promoted cell growth by up-regulating the oncogenic miR-17-92 microRNAs.

指導教授推薦書………………………………………………………....
口試委員會審定書………………………………………………………
授權書……………………………………………………………… iii
誌謝………………………………………………………………… iv
中文摘要…………………………………………………………… v
英文摘要…………………………………………………………... viii
目錄………………………………………………………………... x
第一章 緒論………………………………………………………. 1
1.1 Epigenetic………………………………………………... 1
1.2 Nucleosome……………………………………………… 2
1.3 post-translational histone modifications (PTHMs)……… 3
1.3-1 Histone methylation………………………………. 3
1.4 Lysine-specific demethylase (LSD1)……………………. 4
1.5 microRNA……………………………………………….. 6
1.5-1 miR-17-92 cluster…………………………………... 7
1.6 c-myc…………………………………………………….. 8
第二章 研究動機與目的………………………………………... 9
第三章 材料與方法……………………………………………. 10
3.1細胞株培養………………………………………………. 10
3.2 siRNA 轉染作用 (transfection)………………………… 10
3.3 RNA萃取……………………………………………….. 11
3.4 反轉錄聚合酶連鎖反應(reverse transcription-polymerase chain reaction，RT-PCR)…………………………………….. 11
3.5即時定量聚合酶連鎖反應 (Quantitative-polymerase chain reaction PCR， Q-PCR)……………………………………... 12
3.6 DAPI cell proliferation assay……………………………... 12
3.7 mRNA stability assay……………………………………... 13
3.8 Protein stability assay……………………………………... 13
3.9 LSD1 inhibitor (2-PCPA)…………………………………. 14
3.10蛋白質萃取………………………………………………. 14
3.11蛋白質濃度測定…………………………………………. 15
3.12西方墨點分析法…………………………………………. 15
第四章 實驗結果……………………………………………… 17
4.1 膀胱癌組織中LSＤ1的表現……………………………. 17
4.2 抑制膀胱癌細胞的LSD1對於細胞生物功能的影響…... 17
4.3 膀胱癌組織中microRNAs的差異表現…………………. 18
4.4抑制兩株細胞的LSD1探討microRNA的表現………… 18
4.5抑制LSD1觀察miR-17-92的表現……………………… 19
4.6 抑制c-Myc對於膀胱癌細胞生物功能性的影響……… 20
4.7 抑制膀胱癌細胞的c-Myc觀察miR-17-92的表現…….. 21
4.8 膀胱癌細胞抑制LSD1對c-Myc的影響……………….. 21
4.9 抑制膀胱癌細胞中的c-Myc觀察LSD1表現…………. 22
4.10膀胱癌細胞中的LSD1對於c-Myc RNA的調控……… 22
4.11膀胱癌細胞中的LSD1對於c-Myc 蛋白的調控……….. 23
4.12膀胱癌細胞中LSD1活性對於c-Myc 蛋白的調控……. 24
第五章 討論……………………………………………………… 26
圖表說明…………………………………………………………… 29
參考文獻…………………………………………………………… 55
附錄………………………………………………………………… 58

1. Jaenisch, R. and A. Bird, Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet, 2003. 33 Suppl: p. 245-54.
2. Bird, A., Perceptions of epigenetics. Nature, 2007. 447(7143): p. 396-8.
3. Kampranis, S.C. and P.N. Tsichlis, Histone demethylases and cancer. Adv Cancer Res, 2009. 102: p. 103-69.
4. Cheung, P. and P. Lau, Epigenetic regulation by histone methylation and histone variants. Mol Endocrinol, 2005. 19(3): p. 563-73.
5. Klose, R.J., E.M. Kallin, and Y. Zhang, JmjC-domain-containing proteins and histone demethylation. Nat Rev Genet, 2006. 7(9): p. 715-27.
6. Kornberg, R.D. and Y. Lorch, Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome. Cell, 1999. 98(3): p. 285-94.
7. Woodcock, C.L., Chromatin architecture. Curr Opin Struct Biol, 2006. 16(2): p. 213-20.
8. Draker, R. and P. Cheung, Transcriptional and epigenetic functions of histone variant H2A.Z. Biochem Cell Biol, 2009. 87(1): p. 19-25.
9. Black, B.E. and E.A. Bassett, The histone variant CENP-A and centromere specification. Curr Opin Cell Biol, 2008. 20(1): p. 91-100.
10. Redon, C.E., et al., gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin. Adv Space Res, 2009. 43(8): p. 1171-1178.
11. Kouzarides, T., Chromatin modifications and their function. Cell, 2007. 128(4): p. 693-705.
12. Kusch, T. and J.L. Workman, Histone variants and complexes involved in their exchange. Subcell Biochem, 2007. 41: p. 91-109.
13. Shi, Y., Histone lysine demethylases: emerging roles in development, physiology and disease. Nat Rev Genet, 2007. 8(11): p. 829-33.
14. Grant, S., Targeting histone demethylases in cancer therapy. Clin Cancer Res, 2009. 15(23): p. 7111-3.
15. Shi, Y., et al., Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell, 2004. 119(7): p. 941-53.
16. Seligson, D.B., et al., Global histone modification patterns predict risk of prostate cancer recurrence. Nature, 2005. 435(7046): p. 1262-6.
17. Anand, R. and R. Marmorstein, Structure and mechanism of lysine-specific demethylase enzymes. J Biol Chem, 2007. 282(49): p. 35425-9.
18. Klose, R.J. and Y. Zhang, Regulation of histone methylation by demethylimination and demethylation. Nat Rev Mol Cell Biol, 2007. 8(4): p. 307-18.
19. Forneris, F., et al., LSD1: oxidative chemistry for multifaceted functions in chromatin regulation. Trends Biochem Sci, 2008. 33(4): p. 181-9.
20. Lee, M.G., et al., An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation. Nature, 2005. 437(7057): p. 432-5.
21. Kahl, P., et al., Androgen receptor coactivators lysine-specific histone demethylase 1 and four and a half LIM domain protein 2 predict risk of prostate cancer recurrence. Cancer Res, 2006. 66(23): p. 11341-7.
22. Huang, J., et al., p53 is regulated by the lysine demethylase LSD1. Nature, 2007. 449(7158): p. 105-8.
23. Lin, Y., et al., The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1. EMBO J, 2010. 29(11): p. 1803-16.
24. Sato, F., et al., MicroRNAs and epigenetics. FEBS J, 2011. 278(10): p. 1598-609.
25. Lee, Y., et al., MicroRNA genes are transcribed by RNA polymerase II. EMBO J, 2004. 23(20): p. 4051-60.
26. Gregory, R.I., et al., The Microprocessor complex mediates the genesis of microRNAs. Nature, 2004. 432(7014): p. 235-40.
27. Yi, R., et al., Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev, 2003. 17(24): p. 3011-6.
28. Schwarz, D.S., Y. Tomari, and P.D. Zamore, The RNA-induced silencing complex is a Mg2+-dependent endonuclease. Curr Biol, 2004. 14(9): p. 787-91.
29. Zeng, Y., R. Yi, and B.R. Cullen, MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci U S A, 2003. 100(17): p. 9779-84.
30. Lu, J., et al., MicroRNA expression profiles classify human cancers. Nature, 2005. 435(7043): p. 834-8.
31. Garzon, R., et al., MicroRNA expression and function in cancer. Trends Mol Med, 2006. 12(12): p. 580-7.
32. O'Donnell, K.A., et al., c-Myc-regulated microRNAs modulate E2F1 expression. Nature, 2005. 435(7043): p. 839-43.
33. He, L., et al., A microRNA polycistron as a potential human oncogene. Nature, 2005. 435(7043): p. 828-33.
34. Ventura, A., et al., Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell, 2008. 132(5): p. 875-86.
35. Volinia, S., et al., A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A, 2006. 103(7): p. 2257-61.
36. Ota, A., et al., Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res, 2004. 64(9): p. 3087-95.
37. Tagawa, H., et al., Synergistic action of the microRNA-17 polycistron and Myc in aggressive cancer development. Cancer Sci, 2007. 98(9): p. 1482-90.
38. Yu, Z., et al., A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation. J Cell Biol, 2008. 182(3): p. 509-17.
39. Amente, S., L. Lania, and B. Majello, Epigenetic reprogramming of Myc target genes. Am J Cancer Res, 2011. 1(3): p. 413-418.
40. Han, J., et al., Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell, 2006. 125(5): p. 887-901.
41. Mendell, J.T., miRiad roles for the miR-17-92 cluster in development and disease. Cell, 2008. 133(2): p. 217-22.