肺癌及肝癌分列於全世界癌症相關致死率的第一及第二名。在我們先前的研究中指出，人類的核醣-5-磷酸異構酶 A (RPIA) 在肺癌及肝癌組織切片中的表現皆大於周邊正常組織。RPIA 能夠藉由調控蛋白磷酸酶 2A (PP2A) 及胞外訊號調節激酶訊息路徑 (Extracellular sigmal-regulated kinases signaling, ERK signaling) 調節肝癌細胞增殖與細胞群落形成能力。除此之外，我們想更深入探討 RPIA 在腫瘤細胞中扮演的角色，因此運用免疫沉澱法(Immunoprecipitation) 及液相層析質譜儀 (LC-MS/MS) 分析可能與 RPIA 結合的蛋白質。我們篩選出 stress-70 protein (GRP75)、78 kDa glucose-regulated protein (GRP78) 及其他六個蛋白質可能與 RPIA 發生結合作用，但仍需要更多證據證實蛋白質之間的直接作用，並闡明這些作用會觸發那些細胞反應機制，最終導致癌細胞增殖能力的調控。另外，我們檢視RPIA 與最常見的抑癌基因p53 之間的關係，RPIA在癌細胞中大量表現，若抑制RPIA，p53的表現會增加，伴隨著 p21 的表現增加，p62 的表現降低，說明抑制 RPIA 可能會誘發細胞衰老、自噬作用及p53 相關的途徑以延緩肝癌及肺癌的形成。而我們先前的研究也發現在大腸直腸癌細胞中，抑制 RPIA 會促使磷酸化 GSK3表現量下降及-catenin 被降解。我們在 PLC5 肝癌細胞中的研究也得到相似的結果，證實Wnt/-catenin 訊號路徑的調控也可能參與在 RPIA 對癌細胞增殖能力的調節。我們的研究結果為抑制 RPIA 減緩癌細胞增殖的現象提供了更多相關的分子機制，並有助於 RPIA 相關癌症治療策略的發展。

Lung cancer and liver cancer are the first and the second leading cause of cancer related death from cancers worldwide. Our previous studies indicate the expression of ribose 5-phosphate isomerase A (RPIA) is higher in lung tumor and liver tumor biopsy than normal adjacent tissues. RPIA is able to regulate liver cancer cell proliferation and colony formation ability via modulating protein phosphatase 2A (PP2A) and extracellular signal-regulated kinases (ERK) signaling pathway. We want to further examine the role of RPIA in cancer cells. Therefore, we applied immunoprecipitation and LC-MS/MS analysis to identify the proteins interacting with RPIA. We identified stress-70 protein (GRP75), 78 kDa glucose-regulated protein (GRP78) and other six proteins which are potential proteins to interact with RPIA. It still requires more evidences to confirm the interactions between RPIA and these proteins and further studies to figure out which cellular responses would be triggered by the protein-protein interactions, and finally lead to the regulation of cancer cell proliferation. Moreover, we examied whether RPIA is related to the most common tumor suppressor, p53, and found that p53 is upregulated by knockdown of RPIA. Accompanied with the increased expression of p21 and decreased expression of p62, RPIA knockdown might trigger cellular senescence, autophagy and p53 dependent tumor suppressive pathway to attenuate liver cancer and lung cancer cell proliferation. According to our previous researches, knockdown of RPIA causes reduced phospho-GSK3levels and degradation of -catenin in colorectal cancer cells. We showed the similar results using PLC5 liver cancer cells, suggests that Wnt/-catenin signaling pathway may contribute to the regulation of cancer cell

proliferation by RPIA. Our studies provide more molecular mechanisms associated with the suppressed cell proliferation by knockdown of RPIA in cancer cells. The information may help the development of RPIA related cancer therapeutic strategies.

Contents
Abstract I
中文摘要 III
致謝 IV
Contents V
Introduction 1
Liver cancer 1
PPP pathway and cancer 1
p53 and its role in tumor suppression 3
Cellular senescence 5
Autophagy 6
Crosstalk between glycolysis and the PPP 7
Wnt/-catenin signaling pathway 7
Aims 8
Materials and Methods 10
Cell culture 10
Transient transfection 10
Lentiviral constructs 11
Establish stable cell lines by lentiviral infection 11
Immunoprecipitation 12
Silver staining 12
Protein extraction 13
LC-MS/MS analysis 14
Western blot analysis 15
RNA extraction and real-time quantitative RT-PCR 16
Cell proliferation analysis by MTT assay 17
Statistical analysis 17
Results 18
Identification of RPIA interacting proteins 18
p53 is up-regulated in RPIA knockdown liver cancer cells and lung cancer cells 19
Knockdown of RPIA elevates the expression levels of cellular senescence and autophagy markers in liver cancer cells and lung cancer cells 20
RPIA does not have effect on protein expression levels of glycolytic enzymes 22
Knockdown of RPIA regulates the expression of proteins involved in Wnt/-catenin signaling pathway 22
Discussion 24
Figures 28
Tables 43
Appendix 46
References 51

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