臺灣博碩士論文加值系統

中文摘要
子宮頸癌的形成肇因於高危險性的人類子宮頸癌病毒持續性的感染，其為目前台灣癌症死因中，高發病率與高死亡率的女性癌症。在分子生物層面中，除了遺傳缺失和突變等基因上的改變會促使癌症發生，附基因體的改變亦包含其中。附基因體改變包含DNA甲基化、組蛋白的修飾與微小核醣核酸的調控作用，其能關掉抑癌基因（tumor suppressor gene）表現。先前實驗室研究發現，在子宮頸癌臨床檢體與癌細胞株中，LMX1A基因的啟動子區域有高度甲基化的現象，而在癌細胞株中表現LMX1A能抑制癌細胞侵襲（invasion）與轉移（metastasis）的能力，因此認定LMX1A在子宮頸癌中具有抑癌基因的功能。在本研究中，我們想進一步釐清LMX1A在子宮頸癌中可能受到哪些分子機制影響。
根據初步實驗測試的結果，在單獨以甲基轉移酶DNMT之抑制劑5-aza-2'-deoxycytidine或結合組蛋白去乙醯脢HDAC之抑制劑Trichostatin A共同作用於細胞中，能促使LMX1A基因有回復表現的現象，由此得知DNA甲基化作用和組蛋白修飾皆參與調控LMX1A表現。我們將LMX1A啟動子區域經由網站分析，發現其上具有密集的Sp1結合位。從先前文獻得知人類乳突瘤病毒的E6、E7蛋白會藉由TGF-beta訊息傳遞活化子宮頸癌細胞中Sp1的表現；失調的p53/Sp1調控機制會刺激肺癌細胞中DNMT1表現；在神經膠質瘤細胞中，Sp1會與HDAC1形成複合體並作用到目標基因上，抑制基因表現。這些研究透露Sp1與附基因調控、癌化過程間密切的關聯性。另一方面，也有研究指出組蛋白甲基轉移酶EZH2可能在子宮頸癌形成的過程中扮演重要的調控角色，因此推論Sp1與EZH2可能包含於LMX1A調控機制中。
在實驗結果部分，我們發現表現Sp1後會提高LMX1A啟動子活性，且隨著Sp1濃度增加，LMX1A啟動子活性與Sp1表現呈現正相關。接著我們也藉由定點突變法與報導基因活性測試的結果中得知，LMX1A啟動子上的Sp1結合位突變會降低啟動子活性，顯示Sp1可能調控LMX1A的表現。另一方面，利用RNA干擾的方式，我們建立了三株EZH2穩定低表現的細胞株，但卻沒有偵測到LMX1A表現有明顯回復的現象，進一步分析LMX1A啟動子區域上的甲基化程度，結果則稍受影響。依據目前的實驗結果，我們推測除了先前已知的DNA甲基化作用之外，在子宮頸癌細胞中，Sp1與EZH2可能參與調控LMX1A的表現。而在未來，我們仍需更多的實驗設計釐清其所包含的分子機制。

英文摘要（Abstract）
Cancer has become the leading cause of death in Taiwan, and cervical cancer is the one with the highest incidence and mortality in women. In addition to genetic deletions and mutations, many studies suggested that epigenetic alterations including DNA methylation and histone modification are also involved in inactivation of tumor suppressor genes (TSG). An epigenomic approach used in our previous work showed that LMX1A is methylation-silenced in cervical cancer. LMX1A, a LIM-homeobox gene, is known to participate in developmental event. Furthermore, we characterized the function of LMX1A by examining cell lines, animal models and human cervical neoplastic tissues, and found that tumor-suppressor function of LMX1A in cervical cancer, especially in invasion and metastasis. Hence, we would like to further elucidate what mechanisms are involved in epigenetic silencing of LMX1A in cervical cancers.
According to the preliminary data, LMX1A was re-expressed in cervical cancer cells which were treated either with 5-aza-2'-deoxycytidine (DAC) only or Trichostatin A (TSA) in combination, we speculated that histone modification might be involved in regulation of LMX1A. Further, we identified the promoter region of LMX1A. After bioinformatic analysis, several transcription factor binding sites, including Sp1 are found in the promoter region of LMX1A. One report demonstrated that HPV-16 E6 and E7 oncoproteins activate Sp1 expression through TGF-beta pathway in cervical cancer. Dysregulation of p53/Sp1 control lead to DNA methytransferase-1 (DNMT1) over-expression has been reported in lung cancer. In addition, Sp1 may interact with histone deacetylase 1 (HDAC1), then down-regulate target gene promoter activity in glioma. Moreover, another report indicated enhancer of zeste homolog 2 (EZH2), which is a highly conserved histone methyltransferase, may be an important epigenetic regulator in cervical cancer carcinogenesis. Therefore, we propose that Sp1 or EZH2 might be involved in the regulation of LMX1A expression.
At first, we found Sp1 could enhance LMX1A promoter activity by overexpression of Sp1. Then we use luciferase reporter assay and site-directed mutagenesis to prove Sp1 might be involved in the regulation of LMX1A.
Secondly, we used RNA interfere (RNAi) to knock down human EZH2. We have selected three stable clones, and confirmed the knockdown efficiency by QPCR and western blot. However, the LMX1A expression level was not significantly restored. We also found that the methylation level of LMX1A promoter in siEZH2 stable clones was slightly lower than control. Taken together, these data suggest that Sp1 and EZH2 might be involved in the regulation of LMX1A. In addition to histone modification, our preliminary data showed that DNA methylation may take part in epigenetic silencing of LMX1A in cervical cancer.

總目錄
正文目錄 II
圖目錄 IV
表目錄 VI
附圖目錄 VII
中文摘要 VIII
英文摘要（Abstract） X

 
正文目錄
第一章 緒論 (Introduction) 1
第一節 子宮頸癌的形成與致病機轉 1
第二節 附基因改變與癌症相關性 2
第三節 LMX1A蛋白質 4
第四節 轉錄因子Sp1 6
第五節 組蛋白甲基轉化酶EZH2 9
第六節 研究起源 12
第二章 材料與方法（Materials & Methods） 16
第一節 Sp1密碼區（CDS）與CDH1啟動子之質體構築 16
第二節 細胞處理 29
第三節 在不同處理細胞中LMX1A的表現量 32
第四節 LMX1A啟動子上甲基化程度之測試 40
第三章 結果 (Results) 48
第一節 附基因體修飾作用與LMX1A表現量間之關聯 48
第二節 轉錄因子Sp1與LMX1A調控機制間之關聯 49
第三節 組蛋白甲基轉化酶EZH2與LMX1A調控機制間之關聯 52
第四章 討論 (Discussion) 55
第一節 轉錄因子Sp1對LMX1A之調控機制 55
第二節 組蛋白修飾作用對LMX1A之調控機制 56
第五章 結論 (Conclusion) 59
第六章 參考文獻 (References) 85

 
圖目錄
圖 1 經去甲基化藥物5-aza-2'-deoxycytidine (DAC)與去乙烯蛋白抑制劑Trichostatin A (TSA)處理後細胞中的LMX1A表現量。 61
圖 2 以溫度梯度PCR及膠體回收DNA方式Sp1密碼區片段。 62
圖 3 利用限制酶KpnI及XhoI或SacI剪切挑選可能具有質體的轉型菌株。 63
圖 4 TA-Sp1載體之定序結果。 64
圖 5 pCMV-tag2b-hSp1載體之定序結果。 65
圖 6 以報導基因活性測試各啟動子片段之LMX1A啟動子活性 66
圖 7 以報導基因活性測試偵測外送轉錄因子Sp1對LMX1A啟動子活性之影響。 67
圖 8 比較野生株與突變Sp1轉錄因子結合區之LMX1A啟動子的活性測試。 69
圖 9 以西方墨點法檢驗細胞株中EZH2的表現量。 70
圖 10 下調EZH2對LMX1A表現量之影響。 71
圖 11 下調EZH2對CDH1表現量之影響。 73
圖 12 構築pGL4.21-CDH1啟動子之載體。 74
圖 13 pGL4.21-CDH1啟動子載體之定序結果。 75
圖 14 下調EZH2對LMX1A啟動子活性之影響。 76
圖 15 以甲基化聚合酵素鏈鎖反應偵測下調EZH2細胞中LMX1A啟動子之甲基化程度。 77
圖 16 以亞硫酸鹽定序確認下調EZH2細胞株內LMX1A啟動子之甲基化程度。 78
 
表目錄
表 1 本研究中所使用之引子對 79
 
附圖目錄
附圖 1 民國98年六月衛生署所公布癌症死因比較圖 80
附圖 2 Sp1與其家族蛋白Sp3之蛋白質結構 81
附圖 3 PRC2複合體的組成因子與EZH2的蛋白結構 82
附圖 4 構築Sp1密碼區及CDH1啟動子所需之TA載體 83
附圖 5 構築Sp1密碼區所用之表現載體pCMV-Tag2(B)載體 83
附圖 6 構築CDH1啟動子所用之報導基因載體pGL4.21 84
附圖 7 轉染shRNA EZH2所用之載體pGIPZ 84
 

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