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研究生:李劭筠
研究生(外文):Shao Yun Li
論文名稱:離胺酸特異性去甲基化酶透過c-Myc調控miR-17-92 群簇表現影響膀胱癌細胞的生長
論文名稱(外文):LSD1 control bladder cancer cell growth through c-Myc regulated miR-17-92 cluster
指導教授:陳淑貞陳淑貞引用關係
指導教授(外文):S. J. Chen
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
論文頁數:72
中文關鍵詞:離胺酸特異性去甲基化酶
外文關鍵詞:LSD1c-MycmiR-17-92 cluster
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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

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