臺灣博碩士論文加值系統

在生物體中為了保持上皮組織的完整性及維持單層上皮細胞的恆定性有兩個假說：contact inhibition所造成的生長停止和擁擠所造成的cell extrusion，目前研究有報導指出這倆個機制的失調會造成多層細胞團塊的產生。在這篇研究我們利用可由IPTG所誘導致癌基因Ha-RasV12表現的MK4細胞發現有多層細胞團塊的產生，而在近期研究發現Hippo-YAP pathway在調控contact inhibition中扮演很重要的角色，在這篇研究我們發現Ha-RasV12會促使YAP入核及表現YAP的下游基因，但是在Ha-RasV12的促使下也會造成LATS1和MST1的表現，暗示著我們Hippo pathway不參與在Ha-RasV12所促使的YAP活化，接著我們發現抑制p-ERK或actomyosin cytoskeleton可以阻止Ha-RasV12所促使的YAP激活，使用了YAP的抑制劑卻無法阻止Ha-RasV12所促使多層細胞團塊的產生，暗示著我們可能有其他分子的參與，Cav1的過度表達可以抑制Ha-RasV12所促使的細胞癌化，因此我們發現Cav1的過度表達也可以抑制Ha-RasV12所促使的多層細胞團塊的產生及YAP活化，然而Cav1的基因敲落只造成YAP激活而沒有產生多層細胞團塊，而跟cell extrusion有關的蛋白Rac和RhoA在Ha-RasV12的誘導下而上升，使用Rac的抑制劑可以阻止Ha-RasV12所促使多層細胞團塊的產生相反地ROCK的抑制劑在正常的細胞會造成多層細胞團塊，抑制p-ERK及Cav1的過度表達也可以阻止Ha-RasV12所促使的Rac活化，最後也發現Rac的過度表達在正常細胞會造成多層細胞團塊，總結來說Ha-RasV12所促使的YAP激活不參與在多層細胞團塊的過程，另一方面Cav1的下調及Rac的活化在多層細胞團塊的行程過程中非常重要。

Two mechanisms are proposed to maintain homeostasis in epithelial cells monolayer: growth arrest caused by contact inhibition and cell extrusion caused by crowding induced delamination. It is highly likely that perturbation of these homeostatic systems lead to the formation of epidermal masses in cancer development. In this study, we showed that IPTG-inducible expression of oncogenic Ha-RasV12 in MK4 cells (a clone of MDCK cells) causes the formation of multilayer cellular aggregation. The Hippo-YAP pathway has recently been shown to be an important regulator of contact inhibition. Induction of Ha-RasV12 triggered YAP nuclear translocation and subsequently YAP-targeted gene expression. Expression of Ha-RasV12 induced elevation of LATS1 and MST1, suggesting that canonical Hippo pathway may not be the cause of Ha-RasV12-induced activation of YAP. Next, we showed inhibition of p-ERK or actomyosin cytoskeleton impeded Ha-RasV12-induced activation of YAP. Treatment of Verteporfin (VP), a YAP/TEAD binding inhibitor, failed to abolish Ha-RasV12 induced multilayer cellular aggregates, indicating the involvement of other molecules. Overexpression of Cav1 inhibited Ha-RasV12 induced cellular and mechanical transformation. In addition, we showed that overexpression of Cav1 inhibited Ha-RasV12-induced YAP activation and multicellular cell aggregates. However, knockdown of Cav1 in MDCK cells only resulted in activation of YAP, but not cellular aggregates. Moreover, Rac and RhoA, both associated with cell extrusion, were increased in Ha-RasV12 overexpressed MK4 cells. EHT1864 (Rac inhibitor) abolished multilayer cellular aggregates in Ha-RasV12-overexpressed MK4 cells, whereas Y27632 (ROCK inhibitor) induced multilayer cellular aggregates in MK4 cells. Inhibition of p-ERK and overexpression of Cav1 also inhibited Ha-RasV12 induced Rac activation. Furthermore, overexpression of Rac1 resulted in cellular aggregates. Taken together, these data indicate that Ha-RasV12-induced YAP activation is not required for multicellular aggregates. On the other hand, Rac activation is essential for Ha-RasV12-induced multilayer cellular aggregate.

Abstract…………………………………………………..…I

中文摘要…………………………………………………………II

致謝……………………………………………………………III

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Introduction…………….………...……………………………1

Material and Methods…………….………………………………...…8

Results…………….………...…………………...…12

Discussion………….………...……………...…….....19

References…….………...…………………………..…….....23

Figure legends...……………...…………………………......29

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