流行病學調查發現, 增加大豆食品的攝取可以減少上皮細胞癌的發生。我們先前的研究證實, 尿液中的大豆異黃酮代謝物可以抑制膀胱癌。但至目前為止，大豆異黃酮抑制癌症的分子機轉並不清楚。因此，我們在抑制膀胱癌細胞生長的情況下，利用subtractive suppression hybridization (SSH)方法尋找表現量不同的基因。從71個基因中篩選出2個候選基因:integral membrane protein 1 (ITM1)及epithelial membrane protein 2 (EMP2) 。利用北方墨點法，我們可以確認, 這兩個基因在給予大豆異黄酮後，的確可以增加其表現量。我們也篩選此二基因在人體多個組織膜中的表現情形。我們發現, EMP2在心臟、胎盤及肺表現量最高;而 ITM1在心臟、胎盤及甲狀腺表現量最高，在胎兒組織中也都有表現。進一步觀察，發現EMP2在不同癌化程度的膀胱癌細胞的表現量不同，而且有隨著癌化程度愈嚴重其表現量愈低的趨勢。為了要更進一步了解大豆異黄酮是如何調控EMP2的表現，因此我們構築了EMP2的啓動子以進一步探討。首先，我們構築了-1420到+268 bp啓動子，亦將啓動子依序截短，嘗試找出大豆異黄酮調控的位置(from -865, -333, -220, -167, -120, -72, -44, -24, and -8 bp to +268 bp) 。我們發現，當啓動子被截短到-24 bp時，其啓動子活性會大幅降低。因此，它的啓動子核心應該在轉錄起點5'端33 bp。細胞在給予大豆異黃酮後，可以增加啓動子活性1.5至2.6倍，而其主要調控點應該是Sp1的結合位置。然而在沒有血清的情況下進行細胞培養，其啓動子活性亦增加1.5至3倍。為了評估EMP2在細胞層次的影響，我們將EMP2接到會發綠色螢光的質體GFP，送入膀胱癌細胞TSGH8301內，挑選持續表現的細胞株。由此我們發現，EMP2位在細胞膜及內質網上，且會使細胞膜起皰。活體外試驗(In vitro)證實在EMP2大量表現下，細胞生長速率並沒有影響，但卻可以降低細胞轉型的能力，增加細胞移動速率。在老鼠動物實驗中也發現，大量表現EMP2細胞其生成的腫瘤較小。 由此研究發現，大豆異黃酮藉由調控啓動子增加EMP2的表現量，而不論在體外或體內實驗，皆證實EMP2在膀胱癌具有抑制癌症生長的功能。

A number of epidemiological studies have reported that increased soy consumption is associated with a reduced risk of several human epithelial cancers. Soy isoflavones have been demonstrated to have a tumor suppressor effect on bladder cancer. To explore the underlying mechanisms, we exploited subtractive suppression hybridization （SSH）to identify the differentially expressed genes associated with growth arrest of tumor cells. Integral membrane protein 1 (ITM1) and epithelial membrane protein 2 (EMP2) were selected among 71 candidate clones. Northern blot analysis confirmed the up-regulation of above two genes after isoflavone treatment. We first examined the distribution and expression pattern of these two genes by human tissue blot analysis. EMP2 was highly expressed in the heart, placenta and lung; while ITM1 was prominently expressed in the heart, placenta, and thyroid. Both genes were expressed in the fetal tissues. Then expression of EMP2 was assessed in a spectrum of uroepithelial cell lines. There was a trend toward negative correlation between EMP2 expression and histological grading of cancer cells. To clarify the regulatory mechanism, the promoter of EMP2 (from -1420 to +268 bp) and a series of 5’-end deletion fragments (from -865, -333, -220, -167, -120, -72, -44, -24, and -8 bp to +268 bp) were cloned into a luciferase reporter plasmid pGL3-baisc. A dramatically decreased promoter activity was demonstrated if the promoter was deleted to -24 bp. The core promoter region of EMP2 thus was determined at 34 bp upstream to transcription start site. The luciferase repoter analysis showed a 1.5 to 2.6-fold of elevated promoter activity after isoflavone treatment; and 1.5 to 3-fold elevation when serum was depleted from the culture medium. To explore the biological relevance of EMP2, an EMP2-GFP fusion protein was constructed, and then a TSGH8301 bladder cell line stably expressed EMP2-GFP was established. In the stable clone, EMP2-GFP was localized on the plasma membrane and ER. Membrane blebbing was observed in cells with over-expression of EMP2. Over-expression of EMP2 did not alter the growth rate, but seems to decrease the foci formation and anchorage independent growth compared with the control cells. In the animal study, over-expression of EMP2 inhibits the tumorigenicty in vivo. But EMP2 appears to enhance the cell migration. In conclusion, a potential tumor suppressor gene-EMP2 was identified by SSH profiling in a model experiment in vitro. Isoflavones can up-regulate the expression of EMP2 through modulation of its promoter. The significance of EMP2 as a tumor suppressor for bladder cancer was proved by in vitro and in vivo studies.

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