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系統識別號 U0026-1708201410105300
論文名稱(中文) 前胸腺素在肺氣腫中調控miR-223的角色探討
論文名稱(英文) The Role of Prothymosin α in the Regulation of miR-223 Expression in Emphysema
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) Department of Biochemistry and Molecular Biology
學年度 102
學期 2
出版年 103
研究生(中文) 陳逸琪
研究生(英文) Yi-Chi Chen
學號 S16011031
學位類別 碩士
語文別 中文
論文頁數 67頁
口試委員 指導教授-吳昭良
口試委員-蕭璦莉
口試委員-陳玉玲
口試委員-曾堯麟
口試委員-張孟雅
中文關鍵字 肺氣腫  前胸腺素  組蛋白去乙醯化酶 2  miR-223 
英文關鍵字 emphysema  ProT  miR-223  HDAC2 
學科別分類
中文摘要 肺氣腫 (Emphysema) 是慢性阻塞性肺疾病 (Chronic Obstructive Pulmonary Disease, COPD) 的主要亞型之一,其病理特徵為肺泡異常擴張、肺泡壁瓦解、喪失肺臟彈性等,最後導致呼吸系統失能。臨床研究發現,病人肺臟的組蛋白去乙醯化酶 2 (histone deacetylase 2, HDAC2) 之表現量及活性有明顯的下降,並且與病情的嚴重程度呈現負相關,但造成 HDAC2 表現量減少的機制仍不清楚。越來越多的研究指出表觀遺傳學 (epigenetics) 與疾病的致病機轉有很大的關係,例如組蛋白乙醯化、DNA 甲基化、訊息核醣核酸的穩定度以及蛋白質的轉譯後修飾。MicroRNA (miRNA) 是一種非編碼的微小 RNA ,可與訊息核醣核酸結合、進而影響訊息核醣核酸之穩定度與功能。文獻報導指出,在肺氣腫檢體中會偵測到高表現量的 MiRNA-223 (miR-223),我們進一步利用生物資訊軟體分析 miR-223 的標定核酸序列,結果發現 miR-223 可以和 HDAC2 的訊息核醣核酸結合,因此我們假設肺氣腫藉由 miR-223 的調控使 HDAC2 表現量下降。前胸腺素 (Prothymosin α, ProT) 是一種酸性核蛋白,參與調節細胞中的許多生理功能,包括染色質重組、細胞增生、免疫與氧化壓力等。前胸腺素過量表現的基因轉殖小鼠會產生自發性的肺氣腫,且實驗證實其 HDAC2 表現量下降,與臨床研究結果相吻合,同時也暗示前胸腺素過量表現可能會導致 miR-223 表現量升高。因此本論文主要在探討前胸腺素、 miR-223 與 HDAC2 之間的關係,藉由此研究提出在臨床上,肺氣腫病患之 HDAC2 下降的可能原因。在細胞實驗中,我們發現前胸腺素過量表現可以透過 NF-κB 的調控,導致 miR-223 表現上升,進而讓 HDAC2 表現降低;另外動物實驗中,前胸腺素過量表現的轉殖肺臟有同樣的現象。最後我們將臨床肺氣腫檢體,利用免疫染色以及原位核酸雜合技術 (in situ hybridization),觀察前胸腺素、HDAC2 和 miR-223 的表現,結果亦與細胞實驗及動物實驗吻合。本論文研究之重要性在於提出了肺氣腫病患之 HDAC2 表現量下降的可能原因。
英文摘要 Emphysema, a major subtype of chronic obstructive pulmonary disease (COPD), results from histone deacetylase 2 (HDAC2) dysfunction and inflammatory responses. However, the underlying mechanism is still unclear. We have previously demonstrated that the expression of HDAC2 is decreased in prothymosin α (ProT) transgenic mice, which exhibit an emphysema-like phenotype. Moreover, microarray data show that expression of miR-223 is increased in ProT transgenic mice, which is consistent with clinical reports. Bioinformatic prediction revealed that HDAC2 mRNA may contain miR-223 target sequences. Here, we proposed that ProT may contribute to the pathogenesis of emphysema by inhibiting HDAC2 expression through upregulating miR-223 expression. We show that miR-223 expression was elevated in ProT transgenic mice and patients with emphysema. HDAC2 expression was decreased in miR-223-overexpressing A549 cells. ProT overexpression decreased HDAC2 expression, whereas treatment with lentiviral vectors expressing the miR-223 target sequence (miR-223 sponge) abrogated the downregulation of HDAC2 expression in ProT-overexpressing cells. Notably, suppression of miR-223 activity reduces progression of emphysema in ProT transgenic mice. Taken together, our results provide a novel mechanism of emphysema pathogenesis whereby ProT inhibits HDAC2 expression through enhancing miR-223 expression.
論文目次 中文摘要 I
英文延伸摘要 III
誌謝 VII
總目錄 VIII
圖目錄 XIII
縮寫 XV
第一章 緒論 1
一、肺氣腫 (Pulmonary Emphysema) 1
二、前胸腺素 (Prothymosin α, ProT) 5
三、組蛋白去乙醯化酶 2 (histone deacetylase 2, HDAC 2) 6
四、微小核醣核酸223 (microRNA-223, miR-223) 8
第二章 研究動機 10
第三章 研究目標 11
第四章 材料與方法 12
一、實驗材料 12
1.質體 12
1-1表現載體 12
1-2 RNA干擾質體 13
2.引子 13
3.microRNA real-time PCR引子 13
4.細胞株 14
5. 抗體 14
5-1. 一級抗體 14
5-2. 二級抗體 14
6.實驗動物 14
7.菌株 15
8.試劑 15
8-1細菌培養液 15
8-2細胞培養相關藥品 16
8-3緩衝液 16
8-3.1蛋白質膠體電泳之緩衝液 16
8-3.2原位雜合技術之緩衝液 18
8-3.3報導基因檢測法之緩衝液 19
9.臨床檢體 19
10.菸萃取液 20
11.NF-κB抑制劑 20
二、實驗方法 20
1.細胞培養 20
2.慢病毒生產 21
2-1. 慢病毒生產 21
2-2. 慢病毒感染A549細胞 21
2-3. 慢病毒濃縮 22
3.RNA萃取 22
4.反轉錄 23
4-1. total RNA之反轉錄 23
4-2. miR-223及snRNA U6之反轉錄 23
5.聚合酶連鎖反應 24
6.即時半定量反轉錄酶聚合連鎖反應 24
7.西方墨點法 25
7-1.蛋白樣品製備 25
7-2.蛋白電泳與免疫轉漬 25
8.動物實驗 26
8-1 取得小鼠染色體DNA 26
8-2. ProT轉殖基因小鼠基因型確認 26
8-3. 菸萃取物誘導的肺氣腫動物模式 27
8-4. 由氣管給予慢病毒至小鼠肺部 27
8-5. 組織固定與包埋 27
8-6. 動物檢體切片染色法 28
8-6-1原位雜合技術 28
8-6-2免疫組織化學染色法 29
9.報導基因偵法 30
10.統計分析 30
第五章 結果 31
一、在A549細胞內過量表現ProT會使miR-223表現量增加 31
二、ProT過量表現會使小鼠肺泡的miR-223表現量增加 31
三、在A549細胞內ProT會調控HDAC2的基因表現量 32
四、在ProT同型合子轉殖小鼠肺臟的HDAC2表現較少 33
五、miR-223會負向調控HDAC2的表現 33
六、ProT透過調控miR-223來減少HDAC2的表現 34
七、ProT同型合子轉殖小鼠肺泡有較多的miR-223,且HDAC2的表現量減少 35
八、菸萃取物誘導之肺氣腫動物模式中,抑制小鼠肺部ProT表現可減緩肺氣腫病情,且miR-223的表現量隨著ProT被抑制而下降 35
九、肺氣腫病患肺臟中miR-223的表現量隨著病情惡化而增加,且與ProT的表現量呈正相關性 36
十、隨著肺氣腫病情的惡化,病患肺臟中miR-223的表現越高,而HDAC2表現會越低 36
十一、建構帶有ProT啟動子的報導基因重組質體 37
十二、菸萃取物及其成份對ProT啟動子活性之影響 38
十三、ProT可能透過NF-κB來增加miR-223啟動子活性 39
第六章、結論 40
第七章、討論 41
參考文獻 46
圖表 51
參考文獻 Adcock, I.M., Ford, P., Ito, K., and Barnes, P.J. (2006). Epigenetics and airways disease. Respir Res 7, 21.
Barnes, P.J. (2006). Reduced histone deacetylase in COPD: clinical implications. Chest 129, 151-155.
Barnes, P.J., Shapiro, S.D., and Pauwels, R.A. (2003). Chronic obstructive pulmonary disease: molecular and cellular mechanisms. Eur Respir J 22, 672-688.
Boutten, A., Goven, D., Boczkowski, J., and Bonay, M. (2010). Oxidative stress targets in pulmonary emphysema: focus on the Nrf2 pathway. Expert Opin Ther Targets 14, 329-346.
Chen, C.Z., Li, L., Lodish, H.F., and Bartel, D.P. (2004). MicroRNAs modulate hematopoietic lineage differentiation. Science 303, 83-86.
Chen Y., Hanaoka M., Droma Y., Chen P., Voelkel N.F., and Kubo K. (2010). Endothelin-1 receptor antagonists prevent the development of pulmonary emphysema in rats. Eur Respir J 35, 904-912.
Chung, K.F. (2001). Cytokines in chronic obstructive pulmonary disease. Eur Respir J Suppl 34, 50s-59s.
Crim, C., Calverley, P.M., Anderson, J.A., Celli, B., Ferguson, G.T., Jenkins, C., Jones, P.W., Willits, L.R., Yates, J.C., and Vestbo, J. (2009). Pneumonia risk in COPD patients receiving inhaled corticosteroids alone or in combination: TORCH study results. Eur Respir J 34, 641-647.
de Ruijter, A.J., van Gennip, A.H., Caron, H.N., Kemp, S., and van Kuilenburg, A.B. (2003). Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J 370, 737-749.
Dorhoi, A., Iannaccone, M., Farinacci, M., Fae, K.C., Schreiber, J., Moura-Alves, P., Nouailles, G., Mollenkopf, H.J., Oberbeck-Muller, D., Jorg, S., et al. (2013). MicroRNA-223 controls susceptibility to tuberculosis by regulating lung neutrophil recruitment. J Clin Invest 123, 4836-4848.
Eschenfeldt, W.H., and Berger, S.L. (1986). The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA. Proc Natl Acad Sci U S A 83, 9403-9407.
Evstafieva, A.G., Belov, G.A., Rubtsov, Y.P., Kalkum, M., Joseph, B., Chichkova, N.V., Sukhacheva, E.A., Bogdanov, A.A., Pettersson, R.F., Agol, V.I., et al. (2003). Apoptosis-related fragmentation, translocation, and properties of human prothymosin alpha. Exp Cell Res 284, 211-223.
Ezzie, M.E., Crawford, M., Cho, J.H., Orellana, R., Zhang, S., Gelinas, R., Batte, K., Yu, L., Nuovo, G., Galas, D., et al. (2012). Gene expression networks in COPD: microRNA and mRNA regulation. Thorax 67, 122-131.
Falk, J.A., Kadiev, S., Criner, G.J., Scharf, S.M., Minai, O.A., and Diaz, P. (2008). Cardiac disease in chronic obstructive pulmonary disease. Proc Am Thorac Soc 5, 543-548.
Fujita, Y., Takeshita, F., Kuwano, K., and Ochiya, T. (2013). RNAi Therapeutic Platforms for Lung Diseases. Pharmaceuticals (Basel) 6, 223-250.
Fulci, V., Scappucci, G., Sebastiani, G.D., Giannitti, C., Franceschini, D., Meloni, F., Colombo, T., Citarella, F., Barnaba, V., Minisola, G., et al. (2010). miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis. Hum Immunol 71, 206-211.
Grishok, A., Pasquinelli, A.E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D.L., Fire, A., Ruvkun, G., and Mello, C.C. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23-34.
Haneklaus, M., Gerlic, M., O'Neill, L.A., and Masters, S.L. (2013). miR-223: infection, inflammation and cancer. J Intern Med 274, 215-226.
Hanrahan, J.P., Tager, I.B., Segal, M.R., Tosteson, T.D., Castile, R.G., Van Vunakis, H., Weiss, S.T., and Speizer, F.E. (1992). The Effect of Maternal Smoking during Pregnancy on Early Infant Lung Function. American Review of Respiratory Disease 145, 1129-1135.
Haritos, A.A., Goodall, G.J., and Horecker, B.L. (1984). Prothymosin alpha: isolation and properties of the major immunoreactive form of thymosin alpha 1 in rat thymus. Proc Natl Acad Sci U S A 81, 1008-1011.
Ioannou, K., Samara, P., Livaniou, E., Derhovanessian, E., and Tsitsilonis, O.E. (2012). Prothymosin alpha: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy. Cancer Immunol Immunother 61, 599-614.
Johnnidis, J.B., Harris, M.H., Wheeler, R.T., Stehling-Sun, S., Lam, M.H., Kirak, O., Brummelkamp, T.R., Fleming, M.D., and Camargo, F.D. (2008). Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature 451, 1125-1129.
Kabesch, M., and Adcock, I.M. (2012). Epigenetics in asthma and COPD. Biochimie 94, 2231-2241.
Karetsou, Z., Kretsovali, A., Murphy, C., Tsolas, O., and Papamarcaki, T. (2002). Prothymosin alpha interacts with the CREB-binding protein and potentiates transcription. EMBO Rep 3, 361-366.
Karetsou, Z., Sandaltzopoulos, R., Frangou-Lazaridis, M., Lai, C.Y., Tsolas, O., Becker, P.B., and Papamarcaki, T. (1998). Prothymosin alpha modulates the interaction of histone H1 with chromatin. Nucleic Acids Res 26, 3111-3118.
Karrasch, S., Holz, O., and Jorres, R.A. (2008). Aging and induced senescence as factors in the pathogenesis of lung emphysema. Respir Med 102, 1215-1230.
Kumar, V., Palermo, R., Talora, C., Campese, A.F., Checquolo, S., Bellavia, D., Tottone, L., Testa, G., Miele, E., Indraccolo, S., et al. (2014). Notch and NF-kB signaling pathways regulate miR-223/FBXW7 axis in T-cell acute lymphoblastic leukemia. Leukemia.
Lee, J., Li, Z., Brower-Sinning, R., and John, B. (2007). Regulatory circuit of human microRNA biogenesis. PLoS Comput Biol 3, e67.
Lim, S., Roche, N., Oliver, B.G., Mattos, W., Barnes, P.J., and Chung, K.F. (2000). Balance of matrix metalloprotease-9 and tissue inhibitor of metalloprotease-1 from alveolar macrophages in cigarette smokers. Regulation by interleukin-10. Am J Respir Crit Care Med 162, 1355-1360.
Makarova, T., Grebenshikov, N., Egorov, C., Vartapetian, A., and Bogdanov, A. (1989). Prothymosin alpha is an evolutionary conserved protein covalently linked to a small RNA. FEBS Lett 257, 247-250.
Mannino, D.M., and Buist, A.S. (2007). Global burden of COPD: risk factors, prevalence, and future trends. Lancet 370, 765-773.
Maritz, G.S. (1997). Maternal nicotine exposure induces microscopic emphysema in neonatal rat lung. Pathophysiology 4, 1-7.
Martinez, F.J., Curtis, J.L., Sciurba, F., Mumford, J., Giardino, N.D., Weinmann, G., Kazerooni, E., Murray, S., Criner, G.J., Sin, D.D., et al. (2007). Sex differences in severe pulmonary emphysema. Am J Respir Crit Care Med 176, 243-252.
Matys, V., Kel-Margoulis, O.V., Fricke, E., Liebich, I., Land, S., Barre-Dirrie, A., Reuter, I., Chekmenev, D., Krull, M., Hornischer, K., et al. (2006). TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res 34, D108-110.
Moschos, S.A., Williams, A.E., Perry, M.M., Birrell, M.A., Belvisi, M.G., and Lindsay, M.A. (2007). Expression profiling in vivo demonstrates rapid changes in lung microRNA levels following lipopolysaccharide-induced inflammation but not in the anti-inflammatory action of glucocorticoids. BMC Genomics 8, 240.
Mosoian, A. (2011). Intracellular and extracellular cytokine-like functions of prothymosin alpha: implications for the development of immunotherapies. Future Med Chem 3, 1199-1208.
Noureddine, H., Gary-Bobo, G., Alifano, M., Marcos, E., Saker, M., Vienney, N., Amsellem, V., Maitre, B., Chaouat, A., Chouaid, C., et al. (2011). Pulmonary artery smooth muscle cell senescence is a pathogenic mechanism for pulmonary hypertension in chronic lung disease. Circ Res 109, 543-553.
Schaefer, J.S., Montufar-Solis, D., Vigneswaran, N., and Klein, J.R. (2011). Selective upregulation of microRNA expression in peripheral blood leukocytes in IL-10-/- mice precedes expression in the colon. J Immunol 187, 5834-5841.
Schreiber, J., Jenner, R.G., Murray, H.L., Gerber, G.K., Gifford, D.K., and Young, R.A. (2006). Coordinated binding of NF-kappaB family members in the response of human cells to lipopolysaccharide. Proc Natl Acad Sci U S A 103, 5899-5904.
Shapiro, S.D. (2000). Evolving concepts in the pathogenesis of chronic obstructive pulmonary disease. Clin Chest Med 21, 621-632.
Spira, A., Beane, J., Pinto-Plata, V., Kadar, A., Liu, G., Shah, V., Celli, B., and Brody, J.S. (2004). Gene expression profiling of human lung tissue from smokers with severe emphysema. Am J Respir Cell Mol Biol 31, 601-610.
Su, B.H., Tseng, Y.L., Shieh, G.S., Chen, Y.C., Shiang, Y.C., Wu, P., Li, K.J., Yen, T.H., Shiau, A.L., and Wu, C.L. (2013). Prothymosin alpha overexpression contributes to the development of pulmonary emphysema. Nat Commun 4, 1906.
Vian, L., Di Carlo, M., Pelosi, E., Fazi, F., Santoro, S., Cerio, A.M., Boe, A., Rotilio, V., Billi, M., Racanicchi, S., et al. (2014). Transcriptional fine-tuning of microRNA-223 levels directs lineage choice of human hematopoietic progenitors. Cell Death Differ 21, 290-301.
Wan, E.S., and Silverman, E.K. (2009). Genetics of COPD and emphysema. Chest 136, 859-866.
Yao, H., and Rahman, I. (2012). Role of histone deacetylase 2 in epigenetics and cellular senescence: implications in lung inflammaging and COPD. Am J Physiol Lung Cell Mol Physiol 303, L557-566.
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