先前研究證明，在老鼠肝臟形成過程中，E-cadeherin的表現必需要有HNF4α。因此，當HNF4α表現下降時將會使得肝臟細胞喪失極性並且增加細胞的移動性與侵犯性。然而，HNF4α在人類肝癌細胞的機轉尚未被釐清。日本研究團隊發現一個新的組蛋白甲基轉移酶 (histone methyltransferase), SMYD3會高度表現於人類大腸直腸癌細胞株與肝癌細胞株，並且發現SMYD3會負調控HNF4α的表現，但尚未以實驗證明此關係。我們假設，或許降低HNF4α的表現會藉由降低E-cadeherin的表現，進而增加肝癌細胞的轉移能力。我們的研究主要是鑑定出在人類肝癌細胞中調控HNF4α的重要轉錄因子。在此，我們試驗將HNF4α調控區域中SMYD3的結合序列刪除後是否會增加HNF4α基因的表現，進而降低肝癌細胞的轉移能力。 但是，實驗結果顯示刪除HNF4α調控區域中SMYD3的結合序列不會增加HNF4α基因的表現，或許SMYD3並不是一個重要的轉錄因子。接著，我們更細微的依序刪除HNF4α調控區域。因此可以找出兩個SP1 結合序列，分別位於:(-243與-192)，一個HNF3α(-258)結合序列和一個Cdx2A結合序列(-226到-192)。實驗結果指出，在HNF4α調控區域 (-192)位置的SP1結合序列有可能是無效的，其真正有效位置是位於-243；同時在HNF4α調控區域(-269到-226)位置的HNF3α與SP1不具有協同調控作用。同時CdX2A似乎真的會抑制HNF4α的表現，或許CdX2A對於HNF4α調控E-cadeherin 的表現會是造成肝癌細胞轉移程度不一的原因之ㄧ。

It has been reported that in mouse hepatogenesis HNF4α is essential for the expression of E-cadherin in the organ formation. Thus if HNF4α is down-regulated the consequence would be the lost of polarity and increase in cell mobility and invasiveness of hepatocyte. Whether HNF4α can regulate metastasis of human hepatoma cell has not been determined yet. It has been reported that in most hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) SMYD3, a histone methyaltranferase, was over expressed. SMYD3 can down-regulate HNF4α. The decreased HNF4αexpression may alter carcinogenesis of HCC through decrease expression of E-cadherin, thus increasing metastatic potential of HCC.
The aim of this study is to characterize several transcription factors (TFs) that are important in regulating HNF4α expression in the HCC. Here, we examined whether deletions of SMYD3 binding motif in the promoter region of HNF4αcould increase the metastasis potential of human hepatoma cell lines. The results of functional assay of SMYD3 on HNF4α regulatory sequence could not confirm the previous study. Therefore SMYD3 appeared not to be a critical factor for HNF4α expression in our study. More detailed serial deletions of HNF4αpromoter allowed us to identify three putative positive regulatory elements and one putative negative regulatory element. We want to identify which transcription factor acts as a positive modulator for the HNF4αexpression.
There are two putative SP1 binding motifs in the HNF4α promoter region at (-243/154) and (-192/154), respectively. The results of functional assays indicated that SP1 binding motif in the (-192/154) promoter region may not be functional. The functional SP1 binding motif is located in the position of -243. Our results also showed that Cdx2A is a negative regulator for HNF4α, thus it may be a potential suppressor for E-cadherin expression, it may also contribute to HCC metastasis.

ENGLISH ABSTRCT ˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙I
中文摘要˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙Ⅱ
CONTENT ˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙Ⅲ
INTRODUCTION ˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙1
MATERIALS & METHODS˙˙˙˙˙˙˙˙˙˙˙˙˙˙6
RESULTS ˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙17
DISCUSSION˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙33
REFERENCES˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙37
APPENDIX˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙39

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