藉由微陣列基因晶片以探討中草藥BP011w對於抑制肺腺癌細胞株爬行及轉移之機制

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研究生：	吳秉翰 Bing-Han Wu
論文名稱：	藉由微陣列基因晶片以探討中草藥BP011w對於抑制肺腺癌細胞株爬行及轉移之機制 In vitro analysis of molecular mechanisms underlying effects of Chinese medicine BP011w to inhibit migration of lung adenocarcinoma
指導教授：	蘇立仁
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 系統生物與生物資訊研究所 Graduate Institute of Systems Biology and Bioinformatics
論文出版年：	2016
畢業學年度：	104
語文別：	中文
論文頁數：	78
中文關鍵詞：	肺癌、CL1-5 、非小細胞肺癌、轉移、中草藥、微陣列基因晶片
外文關鍵詞：	Lung cancer, CL1-5, Non-small cell lung cancer, metastasis, Traditional Chinese Medicines, microarray
相關次數：	點閱：23 下載：0
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肺癌在全世界是最常見且死亡率最高的癌症，在台灣也不例外。許多的病人由於肺腺癌的高轉移性而常常在診斷後被發現已經是癌症晚期。就算病人在診斷後為初期就動手術把腫瘤切除，肺腺癌往往會在數個月後轉移復發並且伴隨著高死亡率，這也是肺癌死亡率居高不下的原因。近年來艾瑞莎(Gefitinib)及得舒緩(Erlotinib)是肺癌最常被使用的標靶藥物，但是兩者都沒有抑制肺腺癌細胞轉移的效果，因此尋找一種全新的藥物來針對抑制肺腺癌的轉移是十分重要的。在我們的研究中，我們想要找到一種中國傳統中藥並且具有抑制肺腺癌細胞爬行及轉移的功能，因為中國傳統中藥相對於化學療法及放射療法有許多的優點，像是低副作用、天然、低毒性、便宜等。在經過細胞學實驗的驗證後，我們發現150 μg/ml濃度的BP011w顯著的抑制CL1-5細胞株的爬行及轉移，經由微陣列基因晶片資料的分析後，我們找到了四個候選基因DYNAP、FGF16、miR-548L及REN。在微陣列基因晶片的資料中，DYNAP、FGF16B及REN在BP011w的藥物處理之後基因為下表現，而MIR-548L則為上表現。DYNAP、FGF16B是和PI3K/AKT路徑相關，FGF16B、REN則是和RAS/ERK路徑相關。最後，我們推測是由於BP011w造成這四個基因表現的變化而導致PI3K/AKT及RAS/ERK的下游訊號被阻擋進而造成抑制肺腺癌細胞株的爬行及轉移。

Lung cancer is one of the most frequently diagnosed cancers and is the major cause of cancer-related death worldwide, as well as in Taiwan. Most patients were diagnosed with advanced-stage lung adenocarcinomas because of its highly metastatic rate. Even if diagnosed at an early stage and surgically removed, lung adenocarcinomas can recurrence and extending to other organs with a high mortality rate after a few months. Recently, Gefitinib and Erlotinib were the most common target therapy for lung cancer, but both of them can’t inhibit the cell migration. Therefore, it is necessary to find a brand new drug against to the metastatic of lung cancer, especially for lung adenocarcinoma. In our study, we want to search a Traditional Chinese Medicines to inhibit cell migration of lung adenocarcinoma because TCMs have lots of advantages including less side effects, naturally, low toxicity, inexpensive than chemotherapy and radiotherapy. After identify by cytological analysis, we found the dosage of 150 μg/ml BP011w could inhibit CL1-5 migration obviously. After analysis the data of microarray, we found 4 candidate genes, DYNAP, FGF16, MIR-548L and REN. In the microarray data, DYNAP, FGF16 and REN were down-regulated; miR-548L was up-regulated after treating by BP011w. DYNAP and MIR-548L were related to PI3K/AKT pathway, FGF16 and REN were related to RAS/ERK pathway. Finally, we surmised that the change of 4 gene expression could block the downstream signal of PI3K/AKT and RAS/ERK due to the treatment of BP011w.

Table of contents

中文摘要    i
Abstract     ii
Table of contents    iii
List of tables    v
List of figures    vi
List of supplements tables    vii
List of supplements figures    viii
1. Introduction    1
1-1. Lung cancer    1
1-2. Pathology and clinical stage classification of lung cancer    2
1-3. Metastasis and therapies of lung adenocarcinomas    3
2. Materials and methods    5
2-1. Cell culture    5
2-2. Aqueous extraction of Traditional Chinese Medicines    6
2-3. Wound healing assay    7
2-4. Boyden chamber migration assay    7
2-5. Cell proliferation assay    8
2-6. Flow cytometric analysis for apoptosis and cell cycle    8
2-7. Protein extraction    9
2-8. Western blot assay    10
2-9. RNA extraction    10
2-10. Microarray hybridization    11
2-11. Data analysis    12
3. Results    13
3-1. Experiment workflow     13
3-2. Cytological analysis     13
3-2-1. TCMs screening    13
3-2-2. Cell proliferation in different drug concentrations    15
3-2-3. Cell morphology and apoptosis analysis    16
3-2-4. Cell cycle analysis by flow cytometry    18
3-2-5. Migration analysis by wound-healing assay    20
3-2-6. Migration analysis by Boyden chamber assay    22
3-2-7. Expression of migration-related proteins by western blot    23
3-3. Bioinformatics analysis    26
3-3-1. Genome-wide expression profiling    26
3-3-2. Differential gene expression analysis    27
3-3-3. Function, pathway and protein-protein interaction    28
3-3-4. Candidate genes which were regulated by BP011w    31
3-3-5. Candidate genes which might inhibit cell migration    36
4. Summary and discussion    39
5. Reference    41
6. Supplements    45

List of tables
Table 1. The percentage of total cells for each phase of the cell cycle (G1, S and G2/M)    20
Table 2. The biological process enriched by 1,106 genes    29
Table 3. The cellular component enriched by 1,106 genes    29
Table 4. The KEGG pathway enriched by 1,106 genes    29
Table 5. The 36 up-regulated candidate genes which might inhibit cell migration    32
Table 6. The 67 down-regulated candidate genes which might inhibit cell migration    33

 
List of figures
Figure 1. Flow diagram of the aqueous extraction    6
Figure 2. Screening of 10 potential TCMs for inhibitory capability on CL1-5 cells by wound healing assay    14
Figure 3. Effect of crude BP011w extracts on the cell proliferation.    15
Figure 4. Effect of crude BP011w extracts on the cell morphology and cell apoptosis    16
Figure 5. Cell cycle percentage of crude BP011w extracts by the cell cytometry    19
Figure 6. BP011w inhibited migration of CL1-5 cells according to the would healing assay .    21
Figure 7. BP011w inhibited migration of CL1-5 cells in the Boyden chamber assay    22
Figure 8. Western blot analysis of CL1-5 treated with different concentrations of BP011w for different time conditions    24
Figure 9. The protein-protein interaction of selected 1,106 genes    30
Figure 10. The flow chat of selecting migration-related genes     31
Figure 11. The heat map of selected 103 genes    35
Figure 12. The gene expression patterns of 4 candidate genes     38

 
List of supplements tables
Table S1. Stage grouping for non-small-cell lung cancer    45
Table S2. Antibodies for western blot assay    46
Table S3. Experiment buffer reagents for cytological analysis and biochemistry laboratory    47
Table S4. RNA QA / QC information    48


 
List of supplements figures
Figure S1. The cause of cancer-related death in 2014 and 2004    49
Figure S2. Lung cancer classification    50
Figure S3. The four population of apoptosis analysis by using flow cytometry    51
Figure S4. RNA quality information for array analysis     52
Figure S5. The cell cycle phase in 12 samples    53
Figure S6. The gene expression pattern in dose- and time dependent manner of 36 up-regulated candidate genes    55
Figure S7. The gene expression pattern in dose- and time dependent manner of 67 down-regulated candidate genes    58
Figure S8. The image of GFP- CL1-5 cell    64



                                

1. Ferlay, J., et al., Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International journal of cancer, 2015. 136(5): p. E359-E386.
2. Siegel, R., et al., Cancer statistics, 2014. CA Cancer J Clin, 2014. 64(1): p. 9-29.
3. Ferlay, J., et al., Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. European journal of cancer, 2013. 49(6): p. 1374-1403.
4. Gridelli, C., et al., Non-small-cell lung cancer. Nat Rev Dis Primers, 2015. 1: p. 15009.
5. Colby, T.V., Wistuba, II, and A. Gazdar, Precursors to pulmonary neoplasia. Adv Anat Pathol, 1998. 5(4): p. 205-15.
6. Travis, W.D., et al., International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. Journal of Thoracic Oncology, 2011. 6(2): p. 244-285.
7. Travis, W.D., E. Brambilla, and G.J. Riely, New pathologic classification of lung cancer: relevance for clinical practice and clinical trials. Journal of clinical oncology, 2013. 31(8): p. 992-1001.
8. Goldstraw, P., et al., The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. Journal of thoracic oncology, 2007. 2(8): p. 706-714.
9. Shepherd, F.A., et al., The International Association for the Study of Lung Cancer lung cancer staging project: proposals regarding the clinical staging of small cell lung cancer in the forthcoming (seventh) edition of the tumor, node, metastasis classification for lung cancer. Journal of Thoracic Oncology, 2007. 2(12): p. 1067-1077.
10. Shivapurkar, N., et al., Apoptosis and lung cancer: a review. Journal of cellular biochemistry, 2003. 88(5): p. 885-898.
11. Shih, Y.-W., et al., α-Chaconine-reduced metastasis involves a PI3K/Akt signaling pathway with downregulation of NF-κB in human lung adenocarcinoma A549 cells. Journal of agricultural and food chemistry, 2007. 55(26): p. 11035-11043.
12. Feld, R., L.V. Rubinstein, and T.H. Weisenberger, Sites of recurrence in resected stage I non-small-cell lung cancer: a guide for future studies. Journal of Clinical Oncology, 1984. 2(12): p. 1352-1358.
13. Hoffman, P.C., A.M. Mauer, and E.E. Vokes, Lung cancer. Lancet, 2000. 355(9202): p. 479-85.
14. Nguyen, D.X., et al., WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis. Cell, 2009. 138(1): p. 51-62.
15. Lindeman, N.I., et al., Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. Journal of Thoracic Oncology, 2013. 8(7): p. 823-859.
16. Huang, H.-L., et al., Discovery of prognostic biomarkers for predicting lung cancer metastasis using microarray and survival data. BMC bioinformatics, 2015. 16(1): p. 1.
17. Siegel, R., D. Naishadham, and A. Jemal, Cancer statistics, 2013. CA Cancer J Clin, 2013. 63(1): p. 11-30.
18. Chen, Q.-y., et al., Curcumin inhibits proliferation–migration of NSCLC by steering crosstalk between a Wnt signaling pathway and an adherens junction via EGR-1. Molecular BioSystems, 2015. 11(3): p. 859-868.
19. Ling, C.-q., X.-q. Yue, and C. Ling, Three advantages of using traditional Chinese medicine to prevent and treat tumor. Journal of integrative medicine, 2014. 12(4): p. 331-335.
20. Chu, Y.-W., et al., Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. American journal of respiratory cell and molecular biology, 1997. 17(3): p. 353-360.
21. Yang, P.-C., et al., Characterization of the mucin differentiation in human lung adenocarcinoma cell lines. American journal of respiratory cell and molecular biology, 1992. 7: p. 161-161.
22. Bracke, M., F. Van Roy, and M. Mareel, The E-cadherin/catenin complex in invasion and metastasis, in Attempts to Understand Metastasis Formation I. 1996, Springer. p. 123-161.
23. John, A. and G. Tuszynski, The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis. Pathology oncology research, 2001. 7(1): p. 14-23.
24. Kunoh, T., et al., A novel human dynactin-associated protein, dynAP, promotes activation of Akt, and ergosterol-related compounds induce dynAP-dependent apoptosis of human cancer cells. Molecular cancer therapeutics, 2010. 9(11): p. 2934-2942.
25. Katoh, M., FGF (Fibroblast Growth Factor), in Encyclopedia of Signaling Molecules, S. Choi, Editor. 2012, Springer New York: New York, NY. p. 603-607.
26. Antoine, M., et al., Fibroblast growth factor 16 and 18 are expressed in human cardiovascular tissues and induce on endothelial cells migration but not proliferation. Biochemical and biophysical research communications, 2006. 346(1): p. 224-233.
27. Liu, C., et al., microRNA-548l is involved in the migration and invasion of non-small cell lung cancer by targeting the AKT1 signaling pathway. Journal of cancer research and clinical oncology, 2015. 141(3): p. 431-441.
28. YOSHIJI, H., et al., Suppression of renin-angiotensin system attenuates hepatocarcinogenesis via angiogenesis inhibition in rats. Anticancer research, 2005. 25(5): p. 3335-3340.
29. Jain, R., et al., Solanum nigrum: current perspectives on therapeutic properties. Altern Med Rev, 2011. 16(1): p. 78-85.
30. Wang, C.-K., et al., Integrated treatment of aqueous extract of Solanum nigrum-potentiated cisplatin-and doxorubicin-induced cytotoxicity in human hepatocellular carcinoma cells. Evidence-Based Complementary and Alternative Medicine, 2015. 2015.
31. Wang, H.-C., et al., Solanum nigrum Linn. water extract inhibits metastasis in mouse melanoma cells in vitro and in vivo. Journal of agricultural and food chemistry, 2010. 58(22): p. 11913-11923.
32. Lai, Y.-J., et al., Anti-cancer activity of Solanum nigrum (AESN) through suppression of mitochondrial function and epithelial-mesenchymal transition (EMT) in breast cancer cells. Molecules, 2016. 21(5): p. 553.
33. Son, Y.-O., et al., Ripe fruits of Solanum nigrum L. inhibits cell growth and induces apoptosis in MCF-7 cells. Food and Chemical Toxicology, 2003. 41(10): p. 1421-1428.
34. Li, J., et al., Aqueous extract of Solanum nigrum inhibit growth of cervical carcinoma (U14) via modulating immune response of tumor bearing mice and inducing apoptosis of tumor cells. Fitoterapia, 2008. 79(7): p. 548-556.

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