臺灣博碩士論文加值系統

在許多的肺癌案例中，表皮生長因子接受器(EGFR)常常是不正常的表現和不正常的活化；Sox2是負責調節幹細胞自我跟新的一個重要的轉錄因子，並在肺癌中高度的表現；在肺癌中，我們發現利用生長因子(TGF和EGF)可以活化表皮生長因子接受器並誘導Sox2的表現，異位表現表皮生長因子接受器訊息傳遞(EGFR signaling)下游一個重要的蛋白質 - c-Myc，會誘導Sox2的表現；破壞c-Myc則會使Sox2表現量下降，因此我們認為表皮生長因子接受器訊息傳遞所誘導的Sox2表現是藉由c-Myc所調控的；異位表現Sox2會增加肺癌細胞增生及不依賴接觸的生長，破壞Sox2則會降低肺癌細胞的癌化性質；除此之外，我們發現Sox2被破壞的肺癌細胞會走向自噬性細胞死亡(autophagic cell death)，但是異位表現Sox2則會使肺癌細胞抵禦由飢餓所引發的自噬性細胞死亡，這些實驗數據說明在肺癌細胞裡，Sox2會誘導癌細胞更易生長；而在老鼠活體實驗中我們發現，過度表現Sox2會增加肺癌細胞腫瘤的生長，相反地，抑制Sox2則會使的肺癌細胞腫瘤無法生長，根據上述的老鼠活體實驗，我們認為Sox2在活體中可以增加腫瘤的生長能力；根據微陣列技術(cDNA microarray)的資料分析，我們發現Sox2會調控表皮生長因子接受器，而根據西方點墨法實驗，可以證實Sox2會誘導表皮生長因子接受器的表現，因此我們認為在肺癌細胞裡Sox2和表皮生長因子接受器形成一個互相調控的迴圈並促使肺癌細胞的生長；另外，異位表現Sox2會誘導肺癌細胞產生間質細胞型態轉換成上皮細胞的型態(mesenchymal-epithelial trans-differentiation)，並增加癌症細胞黏附基質的能力，所以，Sox2可能在表皮生長因子接受器下游扮演促使肺癌細胞生長的角色，並且在肺癌的醫療上可以當作一種生物標記或是治療標地物。

Aberrant expression and function of epidermal growth factor receptor (EGFR) is reported in most lung cancer cases. Sox2, a core transcription factor regulating self-renewal of stem cells, is highly expressed in lung cancer. We discovered that EGFR and its ligands (TGF-??nand EGF) induce Sox2 expression in lung cancer. Ectopic expression of c-Myc, a key effector of EGFR signaling, induced Sox2 expression; knockdown of c-Myc decreased Sox2 level in lung cancer, suggesting that EGFR induces Sox2 via a c-Myc dependent pathway. Ectopic expression of Sox2 promoted cell proliferation and anchorage-independent cell growth; knockdown of Sox2 attenuated oncogenic properties of lung cancer. In addition, Sox2-silencing induced autophagic death of lung cancer; overexpression of Sox2 prevented cell from starvation-induced autophagy. These data demonstrates that Sox2 induces oncogenesis of lung cancer. Overexpression of Sox2 promoted tumor growth in xenograft mouse model; knockdown of Sox2 inhibited tumor formation in vivo. These results support the notion that Sox2 enhances tumorigenesis of lung cancer. Through a cDNA microarray analysis for Sox2 target genes, we identified that Sox2 regulates EGFR. Immunoblotting showed that Sox2 induced EGFR expression, suggesting that an EGFR-Sox2-EGFR positive feedback loop exist in lung cancer. In addition, ectopic expression of Sox2 in lung cancer induces mesenchymal-epithelial trans-differentiation and promotes cell-matrix adhesion. Thus, Sox2 may serve as an important effector of EGFR-mediated oncogenesis and provide a novel prognostic biomarker and therapeutic target for lung cancer.

Abstract-------------------------------------------------5
中文摘要(Abstract of Chinese)----------------------------7

Chapter1：Introduction
1.1 Sox2 and lung development----------------------------9
1.2 Sox2 and cancer-------------------------------------10
1.3 Lung cancer and EGFR--------------------------------11
1.4 c-Myc and oncogenesis-------------------------------12
1.5 EGFR and Sox2---------------------------------------13
1.6 Autophagy and cancer--------------------------------14
1.7 Mesenchymal epithelial trans-differentiation--------15
1.8 Project aim-----------------------------------------16

Chapter2：Materials and methods
2.1 DNA plasmid generation------------------------------18
2.2 Cell culture and establish starvation culture condition---------------------------------------------------------18
2.3 Lentiviral Infection--------------------------------19
2.4 RNA extraction and RT-PCR---------------------------20
2.5 Quantitative Real-Time PCR--------------------------21
2.6 Luciferase reporter assay---------------------------22
2.7 Protein extraction and Western blotting------------ 22
2.8 Colony formation----------------------------------- 25
2.9 WST-1 assay---------------------------------------- 25
2.10 BrdU incorporation assay---------------------------26
2.11 Soft agar assay------------------------------------26
2.12 Immunohistochemistry-------------------------------27
2.13 Transmission electron microscopy (TEM)-------------28
2.14 Acridine orange staining---------------------------28
2.15 Wound healing cell migration assay-----------------28
2.16 Cell-matrix adhesion assay-------------------------29
2.17 Xenograft animal model-----------------------------30
2.18 Statistical analysis-------------------------------30

Chapter3：Result
3.1 EGFR signaling induces Sox2 expression in lung cancer-----------------------------------------------------------31
3.2 c-Myc regulates Sox2 expression---------------------31
3.3 Sox2 controls EGFR expression-----------------------32
3.4 Sox2 promotes growth of lung cancer cells-----------33
3.5 Sox2-silencing induces autophagic cell death of lung cancer cells--------------------------------------------33
3.6 Sox2 increases tumor formation of lung cancer cells-34
3.7 Sox2 induces mesenchymal-epithelial trans-differentiation (MET) of lung cancer cells--------------35
3.8 Sox2 promotes cell matrix adhesion------------------36
3.9 Conclusion------------------------------------------36

Chapter4：Discussion
4.1 Discussion------------------------------------------38

Chapter5：Figure legends
Figure1. Sox2 expression is regulated by EGFR signaling.41
Figure2. c-Myc controls Sox2 expression.----------------43
Figure3. Sox2 regulates EGFR expression.----------------45
Figure4. Ectopic expression of Sox2 promotes lung cancer cell growth---------------------------------------------47
Figure5. Sox2-silencing leads to cell growth arrest.----49
Figure6. Sox2-silencing induces autophagy of lung cancer cells.--------------------------------------------------51
Figure7. Effect of Sox2 on tumor formation.-------------54
Figure8. Sox2 induces mesenchymal-epithelial trans-differentiation (MET) of lung cancer. Sox2-silencing leads to cell growth arrest.----------------------------------60
Figure9. Sox2 promotes cell adhesion.-------------------62
Figure10. A model of Sox2 mediated oncogenesis of lung cancer.-------------------------------------------------64
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