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  • 學位論文

第四型岩藻醣轉酶超量表現對肺癌細胞株醣蛋白的影響

Glycoproteomics of fucosyltransferase IV-overexpressed A549 cell

指導教授 : 陳水田

摘要


蛋白質醣修飾的改變在癌症進程中扮演重要的角色,包括細胞增生,細胞侵犯,細胞轉移…等。我們利用超量表現第四型岩藻醣轉酶(fucosyltransferase IV, FucT4)的肺癌細胞株(A549-FucT4)及其對照組細胞株(A549-Mock)作為實驗的模式細胞,以探討第四型岩藻醣轉酶對於細胞的影響。根據先前研究結果指出,第四型岩藻醣轉酶大量表現使得A549-FucT4細胞株在轉移盤移行實驗(transwell migration assay)、細胞貼附實驗(adhesion assay)、明膠酶譜實驗(zymography)和免疫缺陷型小鼠(SCID mice)體中增殖實驗中皆發現較A549-Mock細胞株表現更惡性。本研究中利用兩種不同方式的嗜醣蛋白親和純化法(lectin affinity enrichment)分離出許多具有岩藻醣修飾之醣蛋白,經由液相層析串連電灑游離飛行式質譜儀(LC/ESI-TOF-MS)鑑定醣蛋白身分。根據比對A549-FucT4及A549-Mock細胞株中的醣蛋白,找出14個具有差異的岩藻醣修飾之蛋白。經過綜合生物分子分析軟體(IPA)運算之後發現這14個醣蛋白分別參與細胞增生,細胞貼附,細胞移動,細胞凋亡癌症相關功能。另外在實驗中我們發現A549-FucT4細胞株的生長速度比A549-Mock細胞株快。因此藉由醣蛋白質體學的實驗結果選出參與細胞增生的醣蛋白HGFR及CD109。結果指出,在不同濃度HGF刺激下或經過不同時間後,A549-FucT4細胞株的HGFR磷酸化程度均小於A549-Mock細胞株。但利用TGF-beta 1 處理細胞後,下游訊息smad3蛋白的磷酸化並無顯著差異。實驗結果讓我們瞭解到第四型岩藻醣轉酶大量表現會對癌症進程中的特定蛋白造成功能上的影響。

並列摘要


Protein glycosylation alterations play an important role in cancer progression, including cell proliferation, cell invasion, and cell metastasis. We use A549-FucT4 cells, which over-expressed fucosyltransferase IV, and A549-Mock cell as a control cell line, as a research model to study the influence of fucosylation in cell biology. Our previous data showed that A549-FucT4 is more malignant than A549-Mock based on the transwell migration assay, adhesion assay, zymography assay, and proliferation of cancer cell lines in SCID mice. We also found that the proliferation rate of A549-FucT4 cells are more rapid than A549-Mock cells. In our study, we used two methods of lectin affinity glycoprotein enrichment to enrich fucosylated proteins, and identified their identities through the LC/ESI-TOF-MS. In comparison to the glycoproteins identified in A549-Mock cells, there were 14 distinctive glycoproteins identified solely in A549-FucT4. Using Ingenuity Pathway Analysis software, we found that these 14 glycoproteins are involved in cell migration, cell proliferation, cell movement, and cell apoptosis. We chose HGFR and CD109 for further study based on our glycoproteomic data. The result indicated that A549-FucT4 cells have less HGFR phosphorylation levels in comparison with A549-Mock cells after treatment with HGF in different doses and different incubation times. But there was no obvious difference in smad3 phosphorylation after TGF-beta 1 treatment between the two cell. We think fucosyltransferase IV overexpression may affect particular proteins which take parts in cancer progression.

參考文獻


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