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研究生：	黃姿瑜 Huang, Tzu-Yu
論文名稱：	探討介質素-6醣化型在透過SOX2調控肺癌細胞轉移及幹細胞特性中的角色 Investigating the Role of Specific Interleukin-6 Glycoforms in Regulating Lung Cancer Metastasis and Stem-like Behavior via SOX2 Expression
指導教授：	蘇五洲 Su, Wu-Chou
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	62
中文關鍵詞：	肺癌、介質素-6 、醣化修飾、SOX-2 高表達
外文關鍵詞：	Lung cancer, Interleukin-6, Glycosylation, SOX-2 high expression
相關次數：	點閱：51 下載：0
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在2017年由衛生福利部所公布之統計資料顯示，肺癌的發病率以及死亡率在常見癌症中排名前幾位；因此，如何改善肺癌的治療是有其重要性的。介質素-6 （Interleukin-6）是一個參與在腫瘤發展以及生物免疫系統中，並且能夠調控許多生物功能的細胞激素。而介質素-6所調節下游傳導途徑包括：JAK / STAT 訊號路徑 (Janus kinase/signal transducers and activators of transcription pathway) 和 MAPK cascade (Mitogen-activated protein kinase cascade) 的活化。在文獻中也指出，介質素-6可能是造成癌症治療中多重抗藥性的因素之一，這是由於被 gp130 / MAPK / STAT3 所調節的三個現象：磷酸化 glycoprotein 過表達、上皮間質間轉化以及幹細胞增生所導致。另外，在先前的文獻中指出這個細胞激素在肺癌中可能透過血液中高分泌量以促進其自分泌，進而導致不良預後和癌症病程的推進。蛋白質轉錄後修飾（Post-transcription-modification）在蛋白質生物功能的形成中占了很重要的地位，本研究中我們著重在探討醣化修飾 (Glycosylation) 這一個種類。醣化修飾的發生率相較於磷酸化更加頻繁，可以透過不同程度的後修飾以及修飾種類來改變蛋白的構型及功能。在我們的研究中主要探討的是：肺癌細胞中介質素-6的醣化狀態的改變與細胞行為和信號傳導間的關係。研究結果顯示，當細胞中的介質素-6處於不同的醣化狀態時，將會導致 SOX-2 的表達量以及 STAT3 和 SRC 的磷酸化程度改變。除此之外，在 AS2-IL6-N73Q 細胞中發現高水平的 SOX-2 蛋白與展現幹細胞特性有所關聯。我們也進一步送入 shRNA 去削弱 SOX-2 的表達，並發現當 SOX-2 表達下降時 AS2-IL6-N73Q 細胞的遷移能力也跟著下降。綜合歸納我們的結果可以知道：當肺癌細胞所擁有之介質素-6帶有特定的N-醣基化變異時，可能會通過增加 SOX-2 的表達而促進肺癌細胞轉移，並且可能會展現出幹細胞特性，進而對藥物治療擁有抗性。

The incidence and the mortality rate of lung cancer are ranked in the top few among common cancers, said in recent records. Therefore, improvement of lung cancer treatment become more critical nowadays. Interleukin 6 (IL-6) is a multifunctional cytokine with important role in tumor development and immunobiology. Also, this cytokine modulates downstream signaling pathways including JAK/STAT pathway (Janus kinase/signal transducers and activators of transcription pathway) and MAPK cascade (Mitogen-activated protein kinase cascade). IL-6 is also a contributing factor for multidrug-resistance in cancer due to the gp130/MAPK/STAT3 axis mediates overexpression of p-glycoprotein, EMT transition and stem cell expansion. In lung cancer, autocrine IL-6 and high serum level of IL-6 have correlation with poor prognosis and progression. Post-transcription-modification (PTM) plays an important role in shaping protein biological functions. In this study, we focus on one of the protein PTM, glycosylation, which occurs more frequently than phosphorylation. Our data demonstrated that alteration on glycosylation status of IL-6 in lung cancer cell would influence the cell behavior and signal transduction. The cells had different status of IL-6 glycosylation resulted in varying expression level of SOX-2 and the phosphorylation level of STAT3 and SRC. Also, the high level of SOX-2 protein was found in AS2-IL6-N73Q cell, suggesting that the cell might perform stem-like property. Also, silence of SOX-2 impaired migration of AS2-IL6-N73Q cell. In conclusion, our results supported that loss of specific N-linked glycosylation on IL-6 might promote cell metastasis of lung cancer mainly through increased SOX-2 expression and would result in drug resistance which considered to be one of the stem-like behavior.

List of Contents

中文摘要－II

Abstract－III

誌謝－IV

List of Contents－VI

Chapter 1: Introduction－1
1-1	Lung cancer in Taiwan－2
1-2	The role of Interleukin-6 (IL-6) in cancer－3
1-3	The relationship between IL-6 signaling axis and lung cancer－4
1-4	Aberrant glycosylation in cancers－4
1-5	The role of stem-like cell behavior in cancers－5
1-6	Rationales－6
1-7	Specific aims－6

Chapter 2: Materials and Methods－7
2-1	Materials－8
2-1-1	List of cells－8
2-1-2	List of primary antibodies－10
2-1-3	List of secondary antibodies－11
2-1-4	List of lentiviral shRNA－11
2-1-5	Primers for RT-PCR－11
2-2	Methods－12
2-2-1	Human NSCLC cell lines and cell culture condition－12
2-2-2	Lentiviral transduction experiments－13
2-2-3	Protein extraction and Western blotting－13
2-2-4	RNA extraction and gel staining－14
2-2-5	Wound-healing migration assay－14
2-2-6	Transwell migration assay－14
2-2-7	Medium collecting and IL-6 enzyme-linked immunosorbent (ELISA) assay－15
2-2-8	Colony formation assay－15
2-2-9	MTT assay－15
2-2-10	Trypan blue staining－16
2-2-11	Study of cancer stemness by Ultra-low Attachment Culture Dish－16
2-2-12	Animal study of mice－16
2-2-13	Statistical analysis－17

Chapter 3: Results－18
3-1	Evaluation of proliferation status of lung cancer cells which produce different IL-6 glycoforms.－19
3-2	IL-6 glycosylation status regulates the levels of IL-6 signaling associated proteins and proteins related to malignancy in lung cancer cell.－19
3-3	Loss of N-linked glycosylation on IL-6 increases the migratory ability of lung cancer cell.－20
3-4	Establishment of SOX-2 knocked down cells for further study－21
3-5	IL-6 N-linked glycosylation resulted in high migratory ability via SOX-2 upregulation－22
3-6	Investigation of the relationship between N-linked glycosylation status and stemness.－23

Chapter 4: Discussion and Conclusion－25
4-1	Discussion－26
4-1-1	Altered N-linked glycosylation status will influence the signal transduction in lung cancer.－26
4-1-2	SOX2-mediated increase in motility is through unknown pathways in lung cancer cell.－27
4-1-3	The altered N-linked glycosylation on IL-6 resulted stem-like behavior and drug resistance of chemotherapy.－28
4-2	Conclusion－29

References－30

Figure legends－35

Figures－39
Figure 1. Evaluation of proliferation status of lung cancer cells which produce different IL-6 glycoforms.－41
Figure 2. Expression levels of IL-6 signaling associated proteins and cancer malignancy related proteins in lung cancer cell.－43
Figure 3. Loss of N-linked glycosylation on IL-6 will increase the migratory ability of lung cancer cell.－45
Figure 4. SOX-2 is stably knocked down in AS2-IL6-N73Q/Q2/Q3 cells by lentiviral transduction.－47
Figure 5. Migratory ability is decreased in AS2-IL6-N73Q/Q2/Q3 cells when SOX-2 knock-down.－48
Figure 6. Stable clone selection of N73Q-shVec and N73Q-shSOX2-1 cells for representative of the difference resulting by SOX-2 downregulation.－50
Figure 7. The tumorigenicity effect of SOX-2 downregulation are estimated by in vivo animal study with stable cell clones of N73Q-shVec and N73Q-shSOX2-1.－52
Figure 8. Evaluation of cancer stem-like property in AS2-IL6 and AS2-IL6-N73Q cell by treatment with Cpd-71, a stem-like cell targeting drug.－54
Figure 9. Inductive level of stem markers in cell with altered N-linked glycosylation status is higher in ultra-low attachment culture.－56
Figure 10. The cells carrying with altered N-linked glycosylation on IL-6 shows resistance in diverse level to drugs used in NSCLC chemotherapy.－58

Appendix－59
Figure S1. Microarray analysis of AS2 cell derivatives carrying with IL-6 glycoforms on SRC downstream gene expression panel.－60
Figure S2. The expressing vector of pLKO.1_208001.－61
Figure S3. The expressing vector of pRc/CMV.－62
                                    

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