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  • 學位論文

藉由定量磷酸化蛋白質體學分析探討MCM2在肺癌細胞中的調控網絡

Quantitative phosphoproteomic analysis uncovers the regulatory networks of minichromosome maintenance protein 2 in lung cancer cells

指導教授 : 阮雪芬
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摘要


微小染色體維持蛋白2(MCM2)是DNA複製的主要調控因子。 MCM2與其它MCM蛋白結合形成六聚體複合物(MCM2-7),並發揮具有解旋酶活性的功能。其功能除了用於DNA解旋,還會限制DNA在每個細胞週期僅複製一次。 MCM2在增殖細胞中表現量高,因此在許多種癌症被廣泛用作生物標誌物。然而,MCM2的分子調控機制在肺癌細胞中研究甚少。在這項研究中,我們用A549 (wild-type p53)和H1299(p53-null)細胞株探討MCM2在肺腺癌扮演的角色。研究結果顯示,在A549細胞中過表達MCM2會促進細胞增殖,而在H1299細胞中抑制MCM2表達則會減少細胞增殖。接著,我們進行定量磷酸化蛋白質體學來揭示在肺癌細胞中受MCM2調控的重要下游基因的網絡。我們在過度表達MCM2的A549細胞中共鑑定出594個磷酸化蛋白及1494個磷酸化位點。這些磷酸化位點中,有164磷酸化蛋白具有顯著差異。此外,在低表達MCM2的H1299細胞中,我們鑑定588個磷酸化蛋白及1599個磷酸化位點。這些磷酸化位點中,有82個磷酸化蛋白有顯著差異。這些有顯著差異的磷酸化蛋白參與了RNA剪接,細胞週期和細胞骨架等功能。在表達MCM2的A549細胞過和低表達MCM2的H1299細胞中,我們發現一個共同被調控的磷酸化位點,即是絲氨酸-99(Ser99),它位在高遷移率族蛋白HMG-Ⅰ/ HMG -Y(HMGA1)上。這表明HMGA1-Ser99對肺癌細胞有著重要的調節作用。我們的結果提供肺癌細胞受MCM2調控的磷酸化蛋白質體,並發現其調控磷酸化網絡。這些研究為肺癌治療提供了新的目標。

並列摘要


Minichromosome maintenance protein 2 (MCM2) is a licensing factor for DNA replication. It interacts with other MCM proteins to comprise MCM2-7 complex, which acts as a helicase for DNA unwinding and limits DNA replication to one round per cell cycle. MCM2 has been widely used as a biomarker for proliferation in many types of cancer. However, the molecular regulation underlying MCM2 in lung cancer cells is poorly understood. In this study, we investigated the role of MCM2 in lung adenocarcinoma A549 (wild-type p53) and H1299 (null p53) cells. MCM2 overexpression increased cell proliferation in A549 cells while silencing MCM2 decreased cell proliferation in H1299 cells. We performed global quantitative phosphoproteomic analysis to uncover the important downstream networks regulated by MCM2 in lung cancer cells. We identified 1484 phosphorylation sites in 593 phosphoproteins of MCM2-overexpressed A549 cells. Of these phosphosites, 110 phosphoproteins were significantly changed in response to MCM2 overexpression. In addition, we identified 1599 phosphorylation sites in 592 phosphoproteins of MCM2-silenced H1299 cells. Of these phosphosites, 57 phosphoproteins were significantly changed in response to MCM2 silencing. The differentially regulated phosphoproteins are involved in biological functions such as RNA splicing, cell cycle and cytoskeleton regulation. Functional study demonstrated that MCM2 overexpression promoted cell migration in A549 cells. Moreover, silencing MCM2 inhibits cell migration and induces cell cycle arrest in H1299 cells. Furthermore, we observed a common phosphorylation change at Ser-99 of high mobility group protein HMG-I/HMG-Y (HMGA1) in both MCM2 overexpression and silencing, indicating an important regulatory effect of Ser-99 HMGA1 on lung cancer cells. The phosphoproteomic profiling of MCM2 in lung cancer cells provides new insight about phosphorylation networks regulated by MCM2 and reveals novel targets for lung cancer therapy.

參考文獻


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