細胞連結在上皮細胞的形態及功能維持上扮演一個很重要的角色。我們之前的研究發現，當致癌基因Ha-RasV12在腎臟上皮細胞過度表現時，會引起細胞連結破壞、細胞軟硬度下降以及細胞變性。同時，我們也發現Ha-RasV12過度表現會造成緊密接合蛋白claudin-1的表現量下降。在文獻中已有許多的研究指出，在不同類型的癌細胞之中都可發現到claudin-1的下降，因此claudin-1除了扮演細胞鍵結的蛋白外，也被建議是抑癌因子。在本論文中，我們想要了解Ha-RasV12所造成的claudin-1表現下降，在Ha-RasV12所造成細胞變軟以及細胞變性的過程中扮演什麼樣的角色。首先，我們先利用小髮夾RNA來干擾claudin-1的表現，以探討是否claudin-1的減少可以造成MDCK上皮細胞變軟以及細胞變性。抑制了claudin-1雖不會造成細胞群塊分離，但會造成MDCK及MK4細胞變軟，而且引起細胞變性。免疫螢光染色的結果顯示其他的細胞鍵結蛋白如 E-cadherin、β-catenin和ZO-1並不會因為claudin-1的下降而改變其分布的位置，但是ZO-1的螢光強度是比較弱且斷續的表現。再者，細胞骨架之中肌動蛋白絲的分布受到claudin-1下降的影響，由原先廣泛分布於細胞的上中下層轉而集中於細胞的中下層位置。另一方面，我們利用基因轉染的技術，將帶有綠色螢光的claudin-1送回腎臟上皮的細胞之中，以探討claudin-1的過度表現是否可抑制Ha-RasV12所造成的細胞變軟以及細胞變性。過度表現的claudin-1可以部分抑制Ha-RasV12所造成的細胞群塊分離，但還是無法抵擋Ha-RasV12所造成的細胞變軟。U0126，MEK1/2的抑制劑，可有效的阻斷Ha-RasV12所引起claudin-1的下降以及分布位置的改變，但無法抑制Ha-RasV12所造成的細胞變軟。以上的結果顯示，抑制claudin-1的表現雖會造細胞變軟及細胞變性，但在Ha-RasV12 所造成的細胞變軟及細胞變性中，除了抑制claudin-1的表現之外，仍需其他的因素共同參與。

Our previous studies showed that induction of Ha-RasV12 resulted in cell-cell junction disruption, cell softening and subsequently transformation in MK4 cells (derived from Madin-Darby canine kidney, MDCK cells). We further found that claudin-1, a “tightening” junction protein, were downregulated in mRNA and protein level upon Ha-RasV12 induction. Downregulation of claudin-1 has been reported in several types of cancer. Claudin-1 was also demonstrated to possess the gastric tumor suppressor activity. We wondered whether the suppression of claudin-1 mediated Ha-RasV12-induced cell softening and transformation. We first investigated whether suppression of claudin-1 by shRNA elicits cell softening and transformation ability in normal epithelial cells (MK4 and MDCK cells). Knockdown of claudin-1 resulted in cell softening and an increase in migration and anchorage independent growth ability. Immunofluorescence staining images showed that claudin-1 knockdown only affect the intensity of ZO-1, but not the localization of β-catenin, E-cadherin and ZO-1. Moreover, claudin-1 knockdown decreased the intensity of junctional actin filaments, especially at apical pole of the cell. We further checked whether overexpression of Green Fluorescence Protein (GFP)-claudin-1 abrogates Ha-RasV12-induced cell junction disruption, cell softening and transformation in MK4 cells. Overexpression of claudin-1 partially stunted Ha-RasV12-induced cell scattering but not cell softening. U0126, the MEK1/2 inhibitors, significantly abolished Ha-RasV12-induced claudin-1 downregulation but not cells softening. Our data supported that knockdown of claudin-1 elicited cell softening and transformation. However, to complete Ha-RasV12-induced cell softening and transformation still needs the cooperation of other Ha-RasV12 activated factors.

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