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研究生:郭政宏
研究生(外文):GUO, JENG-HUNG
論文名稱:香煙煙霧增加蝕骨因子表達並促進肺癌轉移
論文名稱(外文):Cigarette smoke promotes IL-6-dependent lung cancer migration and osteolytic bone metastasis.
指導教授:湯智昕
指導教授(外文):TANG, CHIH-HSIN
口試委員:馮逸卿黃偉謙楊順發謝逸憲
口試委員(外文):FONG, YI-CHINHUANG, WEI-CHIENYANG, SHUN-FAHSIEH, YI-HSIEN
口試日期:2024-06-07
學位類別:博士
校院名稱:中國醫藥大學
系所名稱:生物醫學研究所博士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2024
畢業學年度:112
語文別:英文
論文頁數:83
中文關鍵詞:香菸煙霧肺癌IL-6骨轉移破骨細胞
外文關鍵詞:Cigarette smokeLung cancerIL-6Bone metastasisOsteoclast
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全球癌症相關死亡人口中,肺癌占有很大的比例,而吸菸是其進展和擴散的關鍵重要因素。非小細胞肺癌(NSCLC)是肺癌中診斷率最高的腫瘤類型,約佔確診肺癌患者的85%以上。 據估計,約 30-65% 的轉移性肺癌患者會發生骨轉移。眾所周知,吸菸還會加重骨質疏鬆症和其他骨質流失病症。香煙煙霧中含有許多致癌物質,這些物質會攻擊 DNA 並導致人類肺癌中存在的許多已知基因變化。然而,吸菸與肺癌溶骨性骨轉移(另一種骨質流失症)之間的關係仍不清楚。我們的基因集富集分析(GSEA)顯示,吸菸的肺癌患者骨轉換基因表現量較高。我們的臨床樣本和癌症基因組圖譜(TCGA)資料庫均顯示,患有肺癌骨轉移的吸菸者中溶骨因子IL-6的表現增加。我們的細胞研究表明,香菸煙霧萃取物(CSE)和苯駢[α]芘(B[α]P)能刺激IL-6的產生並促進肺癌細胞遷移。這種煙霧誘導的IL-6依賴性遷移與PI3K、Akt和NF-κB信號通路的活化有關。此外,暴露於香菸煙霧的肺癌細胞釋放的IL-6能刺激破骨細胞的發育。值得注意的是,抑制IL-6可以消除香菸煙霧增強的體內肺癌溶骨性骨轉移。我們的數據顯示,吸菸是溶骨性骨轉移的危險因子。因此,阻斷IL-6可能是治療吸菸肺癌患者溶骨性骨轉移的有效方法。
Lung cancer stands as a major contributor to cancer-related fatalities globally, with cigarette smoke playing a pivotal role in its development and metastasis. Non-small cell lung cancer (NSCLC) is the most diagnostic tumor type of lung cancer, accounting for more than 85% of lung cancer patients who have been diagnosed. It has been estimated that ∼30–65% of patients with metastatic lung cancer will develop bone metastases. Cigarette smoke is also recognized as a risk factor for bone loss disorders like osteoporosis. Cigarette smoke contains many carcinogens which attack DNA and cause the many genetic changes known to exist in human lung cancer. However, the association between cigarette smoke and another bone loss disorder, lung cancer osteolytic bone metastasis, remains largely uncertain. Our Gene Set Enrichment Analysis (GSEA) indicated that smokers among lung cancer patients exhibited higher expression levels of bone turnover gene sets. Both The Cancer Genome Atlas (TCGA) database and our clinic samples demonstrated elevated expression of the osteolytic factor IL-6 in ever-smokers with bone metastasis among lung cancer patients. Our cellular experiments revealed that benzo[α]pyrene (B[α]P) and cigarette smoke extract (CSE) promoted IL-6 production and cell migration in lung cancer. Activation of the PI3K, Akt, and NF-B signaling pathways was involved in cigarette smoke-augmented IL-6-dependent migration. Additionally, cigarette smoke lung cancer-secreted IL-6 promoted osteoclast formation. Importantly, blocking IL-6 abolished cigarette smoke-facilitated lung cancer osteolytic bone metastasis in vivo. Our findings provide evidence that cigarette smoke is a risk factor for osteolytic bone metastasis. Thus, inhibiting IL-6 may be a valuable therapeutic strategy for managing osteolytic bone metastasis in lung cancer patients with smoke.
Contents
中文摘要 I
Abstracts II
Abbreviations IV
Contents VI
Figure Contents X
Appendix Figure Contents XII
Appendix Table Contents XIII
Chapter 1. Introduction 1
1.1 Lung cancer and smoking 2
1.2 Osteolytic bone metastasis 2
1.3 Cigarette smoke and lung cancer metastasis 4
1.4 Aim of study 5
Chapter 2. Materials and Methods 6
2.1 Materials 7
2.2 Methods 9
2.2.1 Cell culture 9
2.2.2 Analyses of databases 10
2.2.3 Ingenuity Pathway Analysis (IPA) 11
2.2.4 Western blot 12
2.2.5 mRNA expression analysis 14
2.2.6 Enzyme-linked immunosorbent assay (ELISA) 15
2.2.7 Migration assay 17
2.2.8 Osteoclast differentiation 18
2.2.9 Luciferase activity 19
2.2.10 Osteolytic bone metastasis animal model 20
2.2.11 Immunohistochemistry (IHC) staining 21
2.2.12 Statistical analysis 21
Chapter 3. Results 22
3.1 The expression of IL-6 is associated with bone metastases from lung cancer in smokers 23
3.2 The PI3K, Akt and NF-B signaling pathways are mediated in cigarette smoke-enhanced IL-6-dependent cell migration 24
3.3 Cigarette smoke lung cancer-secreted IL-6 promotes osteoclastogenesis 25
3.4 Blocking IL-6 abolishes cigarette smoke-induced lung cancer osteolytic bone metastasis 26
Chapter 4. Discussion 27
Chapter 5. Conclusion 32
Chapter 6. Figure Legends 34
Figure 1. Association of cigarette smoke in lung cancer patients and bone turnover and bone metastasis from lung cancer in smokers. 35
Figure 2. B[α]P and CSE enhanced IL-6 expression levels in lung cancer cells. 37
Figure 3. The expression level of IL-6 mRNA were analyzed in H292 cells following exposure to B[α]P and CSE. 38
Figure 4. Level of IL-6 demonstrate a positive relationship with the occurrence of bone metastases in smokers. 39
Figure 5. Cigarette smoke enhances the migration of lung cancer cells. 41
Figure 6. The presence of cigarette smoke promotes the migration of H292 cells exposure to B[α]P and CSE. 42
Figure 7. Knockdown IL-6 expression inhibited cell migration of lung cancer cells. 44
Figure 8. Inhibition of IL-6 expression reduced cell migration in H292 cells. 45
Figure 9. Pathways regulate cigarette smoke-induced IL-6 expression and cell migration in lung cancer. 46
Figure 10. The phosphorylation of p85 and Akt in indicated cells was examined by western blot. 47
Figure 11. Blocking cigarette smoke-facilitated induction in IL-6 expression in lung cancer cells. 48
Figure 12. Inhibition PI3K and AKT reduced mobility of lung cancer cell treated cigarette smoke. 49
Figure 13. NF-B transactivation is involved in cigarette smoke-promoted IL-6-dependent cell migration in lung cancer. 50
Figure 14. NF-κB inhibition reduced IL-6 expression in lung cancer cells. 52
Figure 15. Cells were pretreated with PDTC or transfected with p65 siRNA, and cell migration was examined. 53
Figure 16. H292, H292B[α]P and H292CSE cells were treated siRNA to examine IL-6 mRNA expression levels and cell migration. 54
Figure 17. Luciferase activity of NF-κB in lung cancer cells. 55
Figure 18. The IL-6 promoter containing mutation of NF-κB luciferase activity. 56
Figure 19. Cigarette smoke lung cancer-secreted IL-6 promotes osteoclast formation in RAW 264.7 cells. 57
Figure 20. Cigarette smoke lung cancer CM promotes osteoclast formation markers in RAW 264.7 cells. 59
Figure 21. Cigarette smoke lung cancer-secreted IL-6 promotes osteoclast formation in human primary monocytes. 60
Figure 22. IL-6 antibody inhibits H292B[α]P and H292CSE CM promote osteoclast differentiation in RAW 264.7 cells. 61
Figure 23. Blocking IL-6 inhibits cigarette smoke-induced lung cancer metastasis to bone in vivo. 63
Figure 24. Blocking IL-6 abolishes cigarette smoke-promoted lung cancer osteolytic bone metastasis. 64
Figure 25. IL-6 expression in mice model. 66
Figure 26. TRAP staining in mice legs bone. 68
Figure 27. Schematic diagram summarizing the mechanisms by which cigarette smoke promotes IL-6-dependent lung cancer osteolytic bone metastasis. 69
Appendix Figure 70
Appendix Figure 1. 70
Appendix Figure 2. 71
Appendix Figure 3. 72
Appendix Figure 4. 73
Appendix Figure 5. 74
Appendix Figure 6. 75
Appendix table 76
Appendix Table 1. 76
Appendix Table 2. 77
Chapter 7. References 78

Figure Contents
Figure 1. Association of cigarette smoke in lung cancer patients and bone turnover and bone metastasis from lung cancer in smokers. 35
Figure 2. B[α]P and CSE enhanced IL-6 expression levels in lung cancer cells. 37
Figure 3. The expression level of IL-6 mRNA were analyzed in H292 cells following exposure to B[α]P and CSE. 38
Figure 4. Level of IL-6 demonstrate a positive relationship with the occurrence of bone metastases in smokers. 39
Figure 5. Cigarette smoke enhances the migration of lung cancer cells. 41
Figure 6. The presence of cigarette smoke promotes the migration of H292 cells exposure to B[α]P and CSE. 42
Figure 7. Knockdown IL-6 expression inhibited cell migration of lung cancer cells. 44
Figure 8. Inhibition of IL-6 expression reduced cell migration in H292 cells. 45
Figure 9. Pathways regulate cigarette smoke-induced IL-6 expression and cell migration in lung cancer. 46
Figure 10. The phosphorylation of p85 and Akt in indicated cells was examined by western blot. 47
Figure 11. Blocking cigarette smoke-facilitated induction in IL-6 expression in lung cancer cells. 48
Figure 12. Inhibition PI3K and AKT reduced mobility of lung cancer cell treated cigarette smoke. 49
Figure 13. NF-B transactivation is involved in cigarette smoke-promoted IL-6-dependent cell migration in lung cancer. 50
Figure 14. NF-κB inhibition reduced IL-6 expression in lung cancer cells. 52
Figure 15. Cells were pretreated with PDTC or transfected with p65 siRNA, and cell migration was examined. 53
Figure 16. H292, H292B[α]P and H292CSE cells were treated siRNA to examine IL-6 mRNA expression levels and cell migration. 54
Figure 17. Luciferase activity of NF-κB in lung cancer cells. 55
Figure 18. The IL-6 promoter containing mutation of NF-κB luciferase activity. 56
Figure 19. Cigarette smoke lung cancer-secreted IL-6 promotes osteoclast formation in RAW 264.7 cells. 57
Figure 20. Cigarette smoke lung cancer CM promotes osteoclast formation markers in RAW 264.7 cells. 59
Figure 21. Cigarette smoke lung cancer-secreted IL-6 promotes osteoclast formation in human primary monocytes. 60
Figure 22. IL-6 antibody inhibits H292B[α]P and H292CSE CM promote osteoclast differentiation in RAW 264.7 cells. 61
Figure 23. Blocking IL-6 inhibits cigarette smoke-induced lung cancer metastasis to bone in vivo. 63
Figure 24. Blocking IL-6 abolishes cigarette smoke-promoted lung cancer osteolytic bone metastasis. 64
Figure 25. IL-6 expression in mice model. 66
Figure 26. TRAP staining in mice legs bone. 68
Figure 27. Schematic diagram summarizing the mechanisms by which cigarette smoke promotes IL-6-dependent lung cancer osteolytic bone metastasis. 69


Appendix Figure Contents
Appendix Figure 1. 70
Appendix Figure 2. 71
Appendix Figure 3. 72
Appendix Figure 4. 73
Appendix Figure 5. 74
Appendix Figure 6. 75

Appendix Table Contents
Appendix Table 1. 76
Appendix Table 2. 77


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