臺灣博碩士論文加值系統

p63是腫瘤抑制蛋白p53的家族成員之一。由於使用不同的基因啟動子，ΔNp63異構物缺乏N端轉移活化區之結構，因而被認為在細胞週期與細胞凋亡的調控當中與p53及TAp63扮演相反的角色。研究上發現，ΔNp63在許多種類的人類鱗狀細胞癌有過度表現的情形，其中包括了鼻咽癌(NPC)。然而，ΔNp63的過度表現與鼻咽癌致病過程的關係並不清楚。在此研究中，我們使用過度表現ΔNp63的人類鼻咽癌細胞株NPC-076，嘗試去了解ΔNp63的表現在鼻咽癌細胞的增生及調控細胞週期的過程中可能扮演的角色。我們發現藉由專一性的小干擾RNA(ΔNp63 siRNA)抑制NPC-076細胞中過度表現的ΔNp63，則導致細胞增生的幅度明顯地減少。進一步的研究發現，抑制NPC-076細胞株ΔNp63的表現，會導致p27Kip1與p57Kip2兩種細胞週期依賴酶抑制劑(CKIs)分別在mRNA及蛋白質層次的上調。分析細胞週期，發現抑制ΔNp63的表現促使細胞停滯在G1階段，並導致細胞凋亡。同時，在細胞移動力分析的實驗當中，顯現出ΔNp63表現受到抑制的細胞，其移動力會明顯地降低。我們的發現說明了ΔNp63對於NPC-076細胞週期，以及細胞移動力的調控扮演了重要的角色。同時，ΔNp63在NPC-076腫瘤細胞表現型的維持上也具有重要的影響。

p63 is a member of the tumor suppressor protein p53 family. Due to alternative promoter usage, ΔNp63 isoform lacks the N-terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation and cell growth suppression. ΔNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of ΔNp63 overexpression and NPC pathogenesis has not been fully explored. In this study, we use a ΔNp63 overexpressing human NPC cell line (NPC-076) to explore the possible roles of ΔNp63 in cell proliferation and cell cycle regulation. We found that NPC-076 cell proliferation is greatly suppressed when the overexpressed ΔNp63 is silenced by specific ΔNp63 siRNA. Further studies show that ΔNp63 silencing results in the upregulation of CKIs, including p27kip1 and p57kip2 in both mRNA and protein levels. Cell cycle analysis shows that ΔNp63 silencing also results in increased cell cycle arrest at G1 phase and apoptosis. In a scratch wound cell mobility assay, we also show that cell migration ability was dramatically reduced in ΔNp63 silenced cells. Our findings indicate that ΔNp63 plays important roles in the regulation of NPC-076 cell cycle progression and cell mobility, and may play a pivotal role in the maintenance of NPC-076 tumor cell phenotype.

指導教授推薦書
口試委員會審定書
授權書...................................................iii
誌謝......................................................iv
中文摘要....................................................v
Abstract..................................................vi
Introduction...............................................1
Specific Aims..............................................8
Materials and Methods......................................9
Results...................................................16
Discussion................................................20
Conclusion................................................25
References................................................26
Figures
Functional domain of p53 and p63 protein structure....38
Mechanism of RNA interference.........................39
ΔNp63 is overexpressed in NPC-076 cancer cell line....41
RT-PCR shows that endogenous ΔNp63 of NPC-076 was
effectively silenced by ΔNp63 siRNA...................42
Western blot analysis shows that ΔNp63 siRNA
specifically knockdown the expression of ΔNp63 protein
in NPC-076 cells......................................43
ΔNp63 silencing affects the growth rate of NPC-076
cells.................................................44
Regulation of cell cycle by ΔNp63.....................45
Knockdown of the endogenous ΔNp63 results in
upregulation of CDK Inhibitors p27Kip1 and p57Kip2....46
Knockdown of the endogenous ΔNp63 induces NPC-076 cell
apoptosis.............................................48
Knockdown of ΔNp63 expression inhibits the migration of
the NPC-076 cells.....................................49
Tables
Primers used for RT-PCR...............................51
PCR program...........................................52

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