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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王陸海(Wang Lu-Hai) | |
dc.contributor.author | Yi-Pei Lin | en |
dc.contributor.author | 林宜霈 | zh_TW |
dc.date.accessioned | 2021-06-17T04:36:49Z | - |
dc.date.available | 2021-08-13 | |
dc.date.copyright | 2018-08-13 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-08 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70741 | - |
dc.description.abstract | 大部分肺癌病患在初次確診時都已經是晚期且已經發生轉移(metastasis)。轉移是造成癌症病患復發以及死亡的主要原因,並且復發腫瘤通常展現出抗藥性。在這裡我們藉由流式細胞儀篩選出與血清成份有高度結合以及高轉移能力的細胞株,並發現該細胞株會高表現間隙蛋白30.3 (Connexin 30.3, Gjb4) 。高表現 Gjb4 或抑制 Gjb4 表現會分別增加或減少癌細胞在老鼠模型中的轉移。在臨床檢體中我們發現肺癌組織相較於正常組織會高表現Gjb4 (p=0.0026),並發現使用Gjb4在血液樣本的白細胞層 (blood buffy coat)的表現可以鑑別出一到三期 (p=0.002814) 以及第四期 (p<0.0001) 的肺癌病患。我們也發現 Gjb4 的高表現與肺癌病患的較差預後 (poor prognosis) (p=1.4e-4) 和復發 (recurrence) (p=1.9e-12) 有關。 使用同源老鼠模型,我們發現 Gjb4 會促進腫瘤生長。高分子量的血清樣品中以類胰島素生長因子1 (Insulin-like Growth Factor-1, IGF-1)為主要的生長因子,並能夠透過PKC路徑誘導Gjb4表現上升。我們也發現 Gjb4 會透過MET活化Src,並且通過Src的活化來促進腫瘤球形成能力(sphere-forming ability)以及軟膠集落形成 (soft-agar colony formation)。此外,我們也發現了 Gjb4 透過活化 Src 來增強對於抗癌藥物健擇 (gemcitabine) 和癌妥滅 (etoposide) 的抗藥性 (drug resistance)。抑制Gjb4的表現和Src抑制劑柏萊膜 (Dasatinib)同時使用能夠進一步降低健擇 (gemcitabine)的毒殺癌細胞的存活率。總結,我們的研究顯示出Gjb4具有潛力成為一個作為肺癌診斷 (diagnostic) 和評估預後 (prognostic) 的新穎生物標記 (biomarker), 並且Gjb4也可以作為一個潛在的標靶來改善肺癌病患的治療。 | zh_TW |
dc.description.abstract | Most lung cancer patients are diagnosed late with metastasis, which is the major cause of cancer-related death and recurrent tumors that often exhibit chemoresistance. In the present study, we initially identified gap junction beta-4 protein (Gjb4) to be overexpressed in highly metastatic cancer cells selected by their enhanced binding to serum components. Overexpression or knockdown of Gjb4 increased or decreased lung metastasis of syngeneic mice, respectively. We found that Gjb4 expression was higher in lung tumors than normal tissues (p=0.0026), and Gjb4 levels in blood buffy coat samples showed significant performance in diagnosing stage I-III (p=0.002814) and stage IV (p<0.0001) lung cancer. Moreover, high Gjb4 expression levels were correlated with poor prognosis (p=1.4e-4) and recurrence (p=1.9e-12). Using syngeneic mouse model, we observed that Gjb4 was able to promote tumor growth. High molecular weight serum fraction containing the major growth factor component IGF1 was able to induce Gjb4 via PKC pathway. Gjb4 activated Src signaling via MET, and overexpression of Gjb4 enhanced sphere-forming ability and anchorage-independent growth, which were reversed by inhibition of Src. In addition, we demonstrated that Gjb4-mediated Src activation enhanced chemoresistance of cancer cells toward gemcitabine and etoposide. The combination of Gjb4 knockdown, gemcitabine, and dasatinib further enhanced the inhibition of cancer cell viability. Together, our study has identified Gjb4 as a potential novel diagnostic and prognostic biomarker for lung cancer. Targeting Gjb4 may be exploited as a modality for improving lung cancer therapy. | en |
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dc.description.tableofcontents | 目錄
誌謝 i 中文摘要 iii Abstract iv CHAPTER 1: INTRODUCTION 1 1.1 Lung cancer and metastasis 1 1.2 Gap junction protein family in cancer 2 Chapter 2: MATERIALS AND METHODS 4 2.1 Cell culture 4 2.2 Mouse serum collection, biotinylation, deproteinization, and fractionation 4 2.3 Immunofluorescence staining of biotinylated mouse serum 4 2.4 Tail vein metastasis assay and tumor growth model 5 2.5 Tissue Samples 5 2.6 Sphere formation 6 2.7 Exon array 6 2.8 Immunoprecipitation assay 6 2.9 Amino acid sequence alignment 7 2.10 Scrape loading dye transfer assay 7 2.11 Mass spectrometry 7 2.12 RNA extraction and realtime RT-PCR 8 2.13 Establishment of high serum components-binding sublines 8 2.14 Antibodies and reagents 8 2.15 Western blot 8 2.16 Soft agar colony formation assay 9 2.17 MTS cell viability assay 9 2.18 Tail vein metastasis assay and tumor growth model 9 2.19 IHC staining and analysis 10 2.20 Generation of stable cell lines and plasmid construction 10 2.21 Statistical analysis 10 CHAPTER 3: RESULTS 12 3.1 Lung cancer cells with enhanced binding to serum components display enhanced metastatic ability 12 3.2 Gjb4 is overexpressed in high serum components binding C10F4-S cells 13 3.3 Gjb4 promotes metastasis and is correlated with poor clinical outcomes 13 3.4 Gjb4 is overexpressed in tumors and blood buffy coat samples of lung cancer patients 14 3.5 Gjb4 displays a higher performance than CK19 in distinguishing lung cancer patients from healthy people 15 3.6 The promoting role of Gjb4 in tumor formation 15 3.7 Gjb4 knockdown reduces chemoresistance of lung cancer cells 16 3.8 Gjb4 is inducible by components within high molecular weight serum complexes via PKC pathway and promotes chemoresistance via Src activation 16 3.9 IGF1-IGF1R signaling plays a dominant role in the induction of Gjb4 by high molecular weight serum fraction 17 3.10 Gjb4 activates Src via MET, and Src activation is needed by chemoresistance promoted by Gjb4 18 3.11 Gjb4 promotes sphere formation and anchorage-independent growth via Src activation 20 CHAPTER 4:DISCUSSION 23 ACKNOWLEDGEMENTS 27 CHAPTER 5:REFERENCES 84 圖目錄 Figure 1. The binding of mouse serum components to C10F4 cells. 28 Figure 2. The establishment of high serum components-binding sublines by fluorescence-activated cell sorting (FACS). 29 Figure 3. C10F4-S cells exhibit enhanced metastasis. 30 Figure 4. Flowchart of the expression analysis of C10F4-S cells compared with both C10F4-P and normal lung cells from mouse. 31 Figure 5. Eleven genes encoding cell surface proteins that were overexpressed in C10F4-S cells. 32 Figure 6. Gjb4 promotes metastatic outgrowth. 33 Figure 7. Gjb4 is correlated with poor survival and first progression after surgery of lung cancer patients. 35 Figure 8. Gjb4 expression is elevated in tumors of lung cancer patients. 36 Figure 9. Elevated percentage of high Gjb4 and CK19 levels in blood buffy coat RNA samples of lung cancer patients than healthy people. 37 Figure 10. Gjb4 is overexpressed in blood buffy coat RNA samples of stage I-III and stage IV patients versus healthy people. 38 Figure 11. Gjb4 displays a higher performance than CK19 in distinguishing lung cancer patients from healthy people. 39 Figure 12. The promoting role of Gjb4 in tumor formation. 40 Figure 13. C10F4-S cells exhibit enhanced chemoresistance. 41 Figure 14. Gjb4 knockdown reduces chemoresistance of lung cancer cells. 42 Figure 15. Gjb4 is inducible by high molecular weight serum fraction. 43 Figure 16. Treatment with complete or >100 kDa serum fraction increased phosphorylation of PKCα/β II (T638/641) in C10F4 cells. 44 Figure 17. Treatment with Calphostin C inhibited the Gjb4 induction by Frxn>100 kDa. 45 Figure 18. Knockdown of PKCα inhibited the expression of Gjb4 induced by serum fraction retained by a 100 kDa cutoff (frxn>100 kDa). 46 Figure 19. IGF-1 is the major growth factor in Frxn>100 kDa. 47 Figure 20. Treatment of growth factors increased phosphorylation of PKCα/β II (T638/641) and Gjb4 levels in H1650 cells. 48 Figure 21. Treatment of growth factors increased phosphorylation of PKCα/β II (T638/641) and Gjb4 levels in C10F4 cells. 49 Figure 22. Treatment of IGF1R inhibitor picropodophyllin (PPP) decreased Gjb4 expression induced by >100 kDa serum fraction in H1650 cells. 50 Figure 23. Treatment of IGF1R inhibitor picropodophyllin (PPP) decreased Gjb4 expression induced by >100 kDa serum fraction in C10F4 cells. 51 Figure 24. Gjb4 regulates the phosphorylation of Src and STAT3. 52 Figure 25. Src phosphorylation and Gjb4 levels were elevated by serum or serum fraction>100 kDa. 53 Figure 26. Gjb4 knockdown in H1650 cells inhibited STAT3-Y705 phosphorylation at both sparse cell density and two days after confluence. 54 Figure 27. Gjb4 knockdown in C10F4-S cells inhibited STAT3-Y705 phosphorylation at both sparse cell density and two days after confluence. 55 Figure 28. PTEN did not co-immunoprecipitate with Gjb4. 56 Figure 29. Gjb4 knockdown inhibited the phosphorylation (Y1234/1235) and protein levels of Met. 57 Figure 30. Gjb4 knockdown reduced the protein levels of Met via lysosomal degradation. 58 Figure 31. Treatment of bafilomycin A1 rescued the phosphorylation of MET reduced by Gjb4 knockdown. 59 Figure 32. Gjb4 activates Src via Met. 60 Figure 33. Src activation is needed by Gjb4 to promote chemoresistance. 61 Figure 34. The combinatory treatment of Gjb4 knockdown, dasatinib and gemcitabine to achieve a greater inhibitory effect on H1650 cell viability. 62 Figure 35. C10F4-S cells exhibited cellular traits of pluripotent stem cells. 63 Figure 36. C10F4-S and H1650-S cells displayed higher sphere-forming ability than their parental cells. 64 Figure 37. Expression of Gjb4, Oct4, Klf4 and CD133 was enhanced in C10F4-S spheroids compared to C10F4-P spheroids. 65 Figure 38. Overexpression of Gjb4 increased expression of stem cell markers including Oct4, Klf4 and CD44. 66 Figure 39. Gjb4 regulates the expression of CD44. 67 Figure 41. Gjb4 knockdown decreased soft agar colony formation of C10F4-S cells. 69 Figure 42. Treatment of Src inhibitor dasatinib reversed the sphere formation promoted by Gjb4 overexpression. 70 Figure 43. Inhibition of Src by dasatinib attenuated the promoting effects of Gjb4 overexpression in soft agar colony formation of C10F4 cells. 71 Figure 44. Deproteinization of serum abolished its sphere-promoting activity. 72 Figure 45. Treatment of Calphostin C nullified the promoting effect of Frxn>100 kDa in sphere formation. 73 Figure 46. Low similarity between the cytoplasmic C-terminal amino acid sequences of Gja1 and Gjb4. 74 Figure 47. The absence of dye transfer in H1650 and C10F4-S cells. 75 Figure 48. The model of signaling pathway and functions of Gjb4. 76 表目錄 Table 1. Correlation of Gjb4 expression levels with clinicopathologic features of lung tumors. 77 Table 2. Correlation of Gjb4 and CK19 expression levels in blood buffy coat RNA samples with clinicopathologic features of lung cancer patients. 78 Supplementary Table S1. The list of primers and oligomers used in this study. 79 Supplementary Table S2. Information of antibodies and reagents used in this study. 81 Supplementary Table S3. Secreting proteins enriched or exist exclusively in the eluents of C10F4-S cells incubated with serum fraction>100 kDa. 83 | |
dc.language.iso | en | |
dc.title | Gjb4 為一新穎之肺癌生物標記並透過活化 Src 以促進
轉移以及抗藥性 | zh_TW |
dc.title | Gjb4 Serves as A Novel Biomarker for Lung Cancer and
Promotes Metastasis and Chemoresistance via Src Activation | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 徐欣伶,褚志斌,顏伶汝,莊雙恩 | |
dc.subject.keyword | Gjb4,肺癌,生物標記,轉移,抗藥性, | zh_TW |
dc.subject.keyword | Gjb4,Lung cancer,Biomarker,Metastasis,Chemoresistance, | en |
dc.relation.page | 94 | |
dc.identifier.doi | 10.6342/NTU201802631 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-08 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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ntu-107-1.pdf 目前未授權公開取用 | 4.8 MB | Adobe PDF |
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