中心體蛋白CPAP (Centrosomal P4.1-associated protein)除了參與中心體的功能，同時也扮演共同轉錄活化子(co-transcriptional activator)的角色，可以幫助NF-κB以及STAT5的轉錄活性。在發炎環境下，細胞介素6號(IL6)會透過細胞的表面受器，驅動細胞內的訊息傳遞轉錄活化因子3號(STAT3)，進而促進其下游基因的轉錄表現。根據我們先前的研究顯示，在肝癌細胞中，CPAP會參與活化IL6/STAT3這條訊息調控路徑，但其分子機制尚不清楚。在本論文中，我們發現在IL6刺激之下，CPAP蛋白與STAT3會相互作用。進一步利用共同免疫沉澱反應，以及原位PLA分析，我們找到了CPAP蛋白與STAT3相互作用的位置。此外，我們也發現一個蛋白片段N66，具有dominant-negative調控IL6/STAT3的能力。透過細胞實驗、動物實驗，以及血管新生實驗，我們發現N66可以經由抑制IL6/STAT3，抑制其下游所驅動之基因表現，進而抑制肝癌細胞的生長以及血管新生。總之，我們的研究不只找到CPAP與STAT3相互結合的位置，同時也發現一蛋白片段，可以dominant-negative調控由IL6/STAT3所活化的下游基因表現及其所衍生的生理功能。

SUMMARY

Our previous results indicated that CPAP could promote angiogenesis through increased expression of IL8 and/or VEGF by IL6/STAT3 pathway in HCC. Here, by co-immunoprecipitation assay and in situ proximity ligation assay (PLA), we found that CPAP directly interacts with STAT3 upon IL6 stimulus; and the interacting domain between CPAP and STAT3 has mapped as well. On the other hand, we found a potential peptide - N66, which can dominantly inhibit the IL6-mediated STAT3 activation. HCC cells with N66 expression have a lower ability in cell proliferation and colony formation. Matrigel plug assay and xenograft animal model further support the dominant-negative effect of N66. Taken together, these results suggested that CPAP transcriptionally co-activates STAT3 through direct interacting with STAT3 upon IL6 stimulus; and N66 plays a dominant-negative role to decrease the activation of IL6/STAT3 in HCC.

Keywords: CPAP, IL6, STAT3, HCC.

INTRODUCTION
Centrosomal P4.1-associated protein (CPAP) plays an important role in transcriptional co-activation of NF-κB, STAT3 and STAT5. Our previous results indicated that overexpressed CPAP promotes the malignancies of HCC cells through enhancing the activation of IL6/STAT3 pathway. In this thesis, the effects and underlying molecular mechanism of how CPAP contributes to the increased activation of IL6/STAT3 pathway were under investigation.

MATERIALS AND METHODS
Reporter assay and Western blot analysis were performed to determine the effects of CPAP or N66 in IL6/STAT3 pathway. STAT3-driven reporter assay was studied in Huh7 and Hep3B cells. The interaction domain between CPAP and STAT3 was determined by co-immunoprecipitation and in situ proximal ligation assay (PLA). The cell proliferation ability was analyzed by CCK-8, BrdU incorporation and colony formation assay, as well as xenograft animal experiment. Trans-well assay and plug assay were used to determine the effect of CPAP or N66 in angiogenesis.

RESULTS
CPAP promotes the activation of STAT3 and its downstream effects, such as cell proliferation and angiogenesis, in response to IL6 stimulus. A potential peptide, N66, reduces the effects of IL6/STAT3-mediated cell proliferation and angiogenesis in HCC.

CONCLUSION
Our results suggest that CPAP transcriptionally co-activates STAT3 through direct interaction upon IL6 treatment; and N66 has a dominant-negative effect in decreasing the IL6/STAT3-induced HCC carcinogenesis.

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