第一部份 雌性素受體α透過SP-1蛋白結合至人類腫瘤壞死因子α基因啟動區之SP-1 site進而誘導Hep3B肝癌細胞株凋亡之研究 第二部份 過量表現雌性素受體β調控Hep3B肝癌細胞株之細胞週期探討

Part.1 Hep3B cell apoptosis induced by overexpressed estrogen receptor-α is mediated through the interaction between SP1 protein and SP-1 site on tumor necrosis factor-α gene promotor in a ligand-independent manner Part.2 Investigation of the cell cycle regulation by overexpressed estrogen receptor-β in Hep3B cell line

陳曉豫(Hsiao-Yu Chen)

指導教授 ： 黃志揚

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2005年)

https://doi.org/10.6834/CSMU.2005.00065

摘要

第一部分
肝細胞癌(Hepatocellular carcinoma)是世界最常見的癌症之一，為一嚴重危害人類身體健康的惡性腫瘤。台灣屬於肝癌高罹患率高死亡率的地區。由於肝癌的進程非常地快速，且肝癌的致病機轉一般被認為是多重因素和多重步驟過程的機制，因此，從分子生物醫學的角度尋找有利肝癌治療或診斷的機制，是重要且正積極進行的課題。實驗室先前採用了人類Hep 3B肝癌細胞株，證明雌性素以及其受體α (ERα)中，會透過ligand-independent的方式活化人類腫瘤壞死因子α基因的表現，經由death receptor的路徑，切割活化caspase8及caspase3，進而導致DNA斷裂。在本實驗中，更採用流式細胞儀，進一步驗證了先前的結果。同時並利用promotor部份基因deletion、luciferase assay和西方點墨法等，探究其更詳細的分子機制。發現，當delete掉人類腫瘤壞死因子α (TNF-α) 啟動區及luciferase reporter嵌合之DNA中位於啟動區之任一SP-1 site位置時，則ERα活化luciferase基因表現的活性完全受到抑制。同時，並以co-immunoprecipitation的實驗，進一步證明過量表現ERα確實會與Hep 3B細胞內之SP1蛋白結合。綜合以上結果，我們認為ERα確實可透過與SP1蛋白的吸附而與下游基因的SP-1 site結合，進而活化調控下游基因的表現。

第二部分
目前由許多流行病學統計資料及醫學研究發表中，發現男性罹患肝癌之比例較女性高，除了生活型態等等的差異導致外，荷爾蒙的影響或許是很重要的一個因素，其中，雌性激素經由雌性素受體作用，
被推測可能具肝臟組織保護的作用，而雌性素受體(Estrogen receptor；ER)又有兩種型式，ERα與ERβ，它們在不同細胞中可能扮演相同或相異的角色。在此我們同樣採用人類Hep 3B肝癌細胞株，來觀察雌性素以及其受體β在調控肝癌細胞之細胞週期中所扮演的角色。首先在西方點墨法以及RT-PCR的實驗中，我們發現，過量表現雌性素受體β並同時處理雌性素時，c-myc、cyclin D1以及p27無論是RNA或是蛋白質的表現都受到明顯調控；c-myc以及cyclin D1的蛋白及RNA表現量均受到抑制，相反的p27的蛋白及RNA表現量，則受到促進。當進一步以流式細胞儀和MTT assay分別觀察細胞週期的變化與細胞之增生存活率，我們發現過量表現雌性素受體β並同時處理雌性素時，僅具些微G1 phase arrest的現象，唯獨細胞的增生存活並未受到明顯的抑制。因此我們推測在Hep 3B細胞中採用短暫轉殖的系統中過量表現雌性素受體β，會在雌性素的存在下，經活化p27的基因表現，抑制調控細胞週期蛋白c-myc及cyclin D1的基因表現並造成些微G1 phase arrest的現象。

關鍵字

雌性素受體α ； 雌性素受體β ； 人類腫瘤壞死因子α ； SP1蛋白

並列摘要

Part.1
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. It’s also a serious problem to human healthy in Taiwan due to its high incidence and fatality rates. Because of the rapid progress and multi-factor involvement of hepatocarcinoma, the improvements of diagnosis and the treatment outcome are still needed. Therefore, efforts have been made studying the biological mechanisms of hepatocarcinogenesis to these problems. Our previous studies have indicated that overexpressed wt-hER-α may induce cell apoptosis by increasing the hTNF-α gene expression in a ligand-independent manner, to activate caspase8 and cspase3 via the death receptor signaling pathway, resulting in DNA fragmentation in Hep 3B cells. In the present experiments, we further confirm the result using flow cytometry, and explore the detail mechanism using the promoter partial deletion, luciferase assay and western blotting. The data show that the luciferase activity induced by ERα is totally declined with either sp-1 site of human TNF-α-dependent reporter gene construct. Moreover, overexpressed ERα interacted directly with SP1 protein was also observed by co-immunoprecipitation assay. Thus, we demonstrated that cell apoptosis induced by ERα is mediated through TNF-α gene expression by flow cytometry and the associatation of ER-α with SP1 protien which binds to SP-1 site of TNF-promoter is further identified in Hep 3B cell line.

Part.2
Epidemiological studies and experimental data show that the incidence of HCC in men is more frequently than that in women. The hormone could be one of the most important factor, in addition to the different of living style between men and women. Through estrogen receptors, estrogen may protect the liver from cancers. However, ERα and ERβ, may play different roles in cells. The induction of Hep 3B cell apoptosis by ERα was observed in our lab. Here, we further investigate that estrogen and ERβ regulate the cell cycle in Hep 3B cell line. It is found the overexpressed ERβ plus 17-β-estradiol (10-8 M) decreases the RNA and protein levels of c-myc and cyclin D1, but increases the RNA expression and protein content of p27. However, the results of flow cytometry an MTT assay showed only minor G1 phase arrest without any proliferative/survival rate changes. Taken together, the overexpressed ERβ with transient transfection system regulates the cell cycle/growth proteins in a ligand-dependent manner, which resulting in the minor G1 phase arrest in Hep 3B cells.

並列關鍵字

estrogen receptor-α ； estrogen receptor-β ； tumor necrosis factor-α ； SP1 protein

參考文獻

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