在動物細胞中，Wnt途徑為一高度被保留的訊息傳導途徑，並且參與包括分化、胚胎發展和組織生成等許多重要作用。負責傳遞Wnt信號的重要因子，β-catenin，在Wnt路徑被活性化的情況下轉移到細胞核中與TCF/LEF等轉錄因子進行互動，進而轉錄Wnt途徑的下游基因。此外，β-catenin 也被視為原癌基因（proto-oncogene）的一種，並且參與包括結腸直腸癌（colorectal），子宮頸癌 (cervical)，子宮內膜癌 (endometrial)和皮膚癌等癌症的病變機轉。Chibby（Cby）基因是一個在多數物種的演化過程中被保存的小分子量蛋白質，並且具有拮抗Wnt/β-catenin訊息傳導的作用。我們在先前的研究中發現Cby基因的過度表現能防止β-catenin轉移至細胞核中，並且抑制其下游基因的轉錄活性。有基於此，我們認為Cby具有潛在性的癌化抑制能力。在本研究中，我們首先探討了Cby基因在子宮頸癌細胞株HeLa 及SiHa中的內生表現程度，相較於正常系子宮頸細胞，我們發現Cby基因在HeLa及SiHa細胞中均有mRNA（訊息傳遞RNA）以及蛋白質表現降低的情況。接著我們以腺病毒傳遞Cby以及Cby核定位序列移除(Chibby nuclear localization signal deletion, Cby∆NLS)基因至子宮頸癌細胞株中，以探討Chibby基因對於癌細胞腫瘤形成功能上的影響。我們發現在HeLa及SiHa細胞中，Cby以及Cby∆NLS基因均能抑制細胞增生、移動能力與細胞群落產生能力。藉由免疫螢光顯微分析，我們也觀察到Cby及Cby∆NLS基因傳送能降低Ki-67細胞增殖標定蛋白的表現。此外，我們也發現在HeLa及SiHa細胞中過度表現Cby或CbyΔNLS能誘導細胞凋亡(apoptosis)及造成細胞週期的擾動(perturbation)。最後，我們發現過度表達Cby能降低C-Myc及PCNA等受β-catenin/TCF4所調控的基因表現，而這可能有助於解釋Cby在子宮頸癌細胞中所具有的抗腫瘤特性。綜合以上結果，我們認為Cby在子宮頸癌中能當作一種潛在性的腫瘤抑制基因，並且能幫助發展新穎的抗子宮頸癌療法。

The Wnt signaling pathway is highly conserved and participates in many important cellular functions including differentiation, embryonic development and tissue generations. β-catenin, the central mediator of the Wnt signaling, interacts with the TCF/LEF family of transcription factors in the nucleus and initiates downstream gene transcription. In addition, β-catenin is known as a proto-oncogene implicated in numerous cancers including colorectal, cervical, endometrial and skin cancer. Chibby (Cby) is evolutionarily conserved in many species and acts as a repressor of Wnt/β-catenin signaling. In our previous study, we have established that Cby over-expression attenuated β-catenin translocation to nucleus and its transcriptional activity. Thus, it was hypothesized that Cby may possess potential tumor suppressing capabilities. In the present study, we first explored endogenous Cby expression status in human cervical cancer cells: HeLa and SiHa cell lines. It was observed that Cby mRNA and protein levels were significantly down-regulated in both cancer lines compared with primary cervical cells. We then conducted functional assays of tumorigenicity on both cells using adenoviral-encoded Cby and its NLS (nuclear localization signaling) deleted variant (Cby∆NLS). It was found that gene delivery of Cby or Cby∆NLS inhibited the proliferation, invasiveness, and colony forming in HeLa and SiHa cells. Immunofluorescent analysis revealed that Cby or Cby∆NLS gene transfer reduced the expression of Ki-67, a cell proliferative marker. Furthermore, Cby or Cby∆NLS restoration induced apoptosis and perturbed cell cycle progression in both cervical cancer cells. Finally, Cby over-expression decreases the expression of β-catenin/TCF4 regulated genes such as c-myc and PCNA, which might contributed to the anti-neoplastic mechanism for Cby in cervical cancer cell lines. Our results strongly suggest that Cby may serve as a tumor suppressor gene during cervical carcinogenesis, and may facilitate in creation of new therapeutic methods.

Index

Abstract in Chinese- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I
Abstract in English - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -III

Table of Contents
Introduction - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - pg. 1

Specific Aims - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - pg. 5

Experimental Materials and Methods - - - - - - - - - - - - - - - pg. 6

Results - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - pg. 13

Discussion- - - - - - - -- - - - - - - -- - - - - - - -- - - - - - - -- - - - - pg. 18

Future Perspective - - - - - - - -- - - - - - - -- - - - - - - -- - - - - - pg. 22

Figures and Legends- - - - - - - -- - - - - - - -- - - - - - - -- - - - - pg. 24

References - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -pg. 31

Appendix- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -pg. 34

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