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研究生:林聖晏
研究生(外文):Sheng Yen Lin
論文名稱:探討甲狀腺素對miR-21的調控機制
論文名稱(外文):The mechanism of miR-21 regulation by thyroid hormone
指導教授:林光輝林光輝引用關係
指導教授(外文):K. H. Lin
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:66
中文關鍵詞:甲狀腺素肝癌miR-21
外文關鍵詞:thyroid hormonehepatomamiR-21
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甲狀腺素 (3, 3’, 5-triiodo-L-thyronine, T3)是一可調控細胞生長、發育與分化的重要因子,其受體 (Thyroid hormone receptor, TR)屬於固醇類荷爾蒙受體家族之一,是甲狀腺素依賴型的轉錄因子,而甲狀腺素便是藉由細胞核內的甲狀腺素受體將其訊號傳至細胞內。利用microRNA microarray分析技術研究在過度表現甲狀腺素受體的肝癌細胞株中處理T3的情況下其microRNAs的表現量,其中miR- 21在處理T3的情況下的表現量會比較起未處理T3的過度表現甲狀腺素受體肝癌細胞(HepG2-TRα1#1)來得高。而microRNAs主要藉由與目標蛋白的3’ 未轉譯區上的序列做配對,以達到抑制目標蛋白的轉譯表現。在過度表現甲狀腺素受體阿法(TRα1)的肝癌細胞株中,經由T3 10nM處理72小時後miR-21的表現量分別上升了約4.1倍,類似的調控結果也可以在表現甲狀腺素受體貝他(TRβ1)的細胞株中亦可觀察到,相同地,在pri-miR-21的表現量中,可觀察到pri-miR-21確實也會受到T3的時間和劑量上的影響。其可能的調控機制在promoter assay中,可以發現到T3影響miR-21的上游啟動子部位可能位於-990~-931中,並且可能為T3結合在miR-21上游啟動子而促使miR-21表現量上升。同時利用TargetScan 4.0尋找miR-21目標蛋白,並將此資料庫與oligo-microarray作交集後,發現了共有57個基因包含在內。而藉由RNA和protein表現量觀察後,可知Tiam1基因會受到T3的調控,所以可推測出Tiam1可能為miR-21下游目標蛋白,並且在暫時過度表現miR-21時,Tiam1的蛋白量有下降的情況發生,由此可知,Tiam1確實會受到miR-21的調控。此研究擬更進一步探討miR-21在受T3調控之後的生理意義與其在癌症中扮演的角色。
Thyroid hormone (3, 3’, 5-triiodo-L-thyronine)is an important factor in metabolism, growth and development of animals. T3 can mediate its effect by thyroid hormone receptors(TRs). TRs can increase or decrease levels of gene expression by its transcription factor function. By comparing the difference of microRNA expression with or without T3 stimulation in TR over expression Hepatoma cell line, we find out that there are many microRNAs positively regulated by T3, including has-miR-21. microRNAs can repress target protein expression by binding 3’UTR of target gene. When treatment with 10nM T3 72 hours in HepG2 overexpressed TRα1#1, we can discover that miR-21 expression is higher. Similarly, the same regulation also can be observed in HepG2 overexpressed TRβ1. Besides, the expression of primary transcript miR-21 is dependent on T3 concentrations and time-dependent. By promoter assay, the mechanism of T3 regulation to miR-21 is possibly that TRs bind directly to miR-21 promoter region and increase pri-miR-21. By using online software - Targetscan 4.0 to search for miR-21 target gene, the database shows that there are many predicted genes. Then, we correlate the data with oligonucleotide microarray to search for the genes that are affected by T3 and miR-21. By western blot and quantitative RT-PCR, we focus on Tiam1 that can down-regulated by T3 and possibly is miR-21 target gene. In HEK293 transient over-expressed miR-21, we find out that Tiam1 is down-regulated by over-expressed miR-21.
指導教授推薦書 i
口試委員會審定書 ii
碩士論文授權書 iii
誌謝 (Acknowledgements) iv
目錄 v
中文摘要 (Abstract in Chinese) vii
英文摘要 (Abstract in English) viii
前言 (Introduction) 1
研究動機 (Specific aims) 12
材料與方法 (Materials and Methods) 13
1.細胞培養(Cell Culture)
2.T3-depleted serum (Td)之配製(Td serum Preparation)
3.3, 3’, 5-triiodo-L-thyronine(T3)溶液之配製(T3 Preparation)
4. RNA與蛋白質萃取前之細胞處理(Cell Treatment)
5. 細胞RNA萃取(Cell RNA Extraction)
6. microRNA反轉錄聚合酶連鎖反應
(Reverse Transcriptase PCR of microRNA)
7. microRNA即時定量聚合酶連鎖反應
(quantitative Real-Time PCR of microRNA)
8啟動子序列分析(promoter assay)
9. 預測microRNA目標蛋白(Pedicting microRNA target-protein)
10.反轉錄聚合酶連鎖反應(Reverse-Transcriptase PCR)
11.即時定量聚合酶連鎖反應(quantitative Real-Time PCR)
12. 蛋白質之萃取(Protein Extraction)
13. 蛋白質定量(protein Quantitation)
14. 西方點墨法(Western Blotting)
15. CIP處理(CIP Dephosphorylation)
16. 3‘端未轉譯區域報導基因法(3’UTR Reporter Gene Assay)
17. 細胞遷移實驗(Migration Assay)
18.明膠基質金屬蛋白酵素活性測試(Gelatin-zymography)
實驗結果 (Results) 22
1. 即時定量聚合酶連鎖反應偵測HepG2-TRα1、HepG2-TRβ1、HepG2-neo中miR-21在不同T3濃度和時間的表現量
2. 利用即時聚合酶連鎖反應偵測HepG2-TRα1#1中的primary miR-21的表現量
3. cycloheximide不影響T3對miR-21的調控
4. 偵測T3處理後HepG2-TRα1#1細胞中miR-21的Host gene表現量
5. T3調控HepG2-TRα1#1細胞中miR-21上游-990~-931區域
6. 預測miR-21目標蛋白
7. miR-21預測之目標蛋白-Tiam1的mRNA和protein表現量受T3之調控
8. 利用CIP (alkaline phosphotase)偵測Tiam1具磷酸化現象
9. 在HEK293細胞中表現miR-21會降低Tiam1蛋白表現量
10. 利用西方點墨法偵測Tiam1在不同肝癌細胞株的表現情況
11. 在Hep3B細胞中表現miR-21會降低Tiam1蛋白表現量
12. 利用3’端未轉譯區域報導基因法確認T3與Tiam1 3’UTR的關係
13. 過度表現miR-21會抑制Hep3B以及HEK293細胞的遷移能力
14. Hep3B及HEK293細胞中表現miR-21會減少MMP的表現及活性
15. 利用即時定量聚合酶連鎖反應確認動物實驗中miR-21的表現量
討論 (Discussion) 29
參考資料 (References) 35
附錄 (Figure legends) 41
1. O'Donnell, K.A., et al., c-Myc-regulated microRNAs modulate E2F1 expression. Nature, 2005. 435(7043): p. 839-43.
2. Zhang, J. and M.A. Lazar, The mechanism of action of thyroid hormones. Annu Rev Physiol, 2000. 62: p. 439-66.
3. Yen, P.M., Physiological and molecular basis of thyroid hormone action. Physiol Rev, 2001. 81(3): p. 1097-142.
4. Sap, J., et al., The c-erb-A protein is a high-affinity receptor for thyroid hormone. Nature, 1986. 324(6098): p. 635-40.
5. Dayton, A.I., et al., A human c-erbA oncogene homologue is closely proximal to the chromosome 17 breakpoint in acute promyelocytic leukemia. Proc Natl Acad Sci U S A, 1984. 81(14): p. 4495-9.
6. Lazar, M.A., Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev, 1993. 14(2): p. 184-93.
7. Munoz, A. and J. Bernal, Biological activities of thyroid hormone receptors. Eur J Endocrinol, 1997. 137(5): p. 433-45.
8. Hodin, R.A., et al., Identification of a thyroid hormone receptor that is pituitary-specific. Science, 1989. 244(4900): p. 76-9.
9. Cook, C.B., et al., Expression of thyroid hormone receptor beta 2 in rat hypothalamus. Endocrinology, 1992. 130(2): p. 1077-9.
10. Baniahmad, A., et al., Interaction of human thyroid hormone receptor beta with transcription factor TFIIB may mediate target gene derepression and activation by thyroid hormone. Proc Natl Acad Sci U S A, 1993. 90(19): p. 8832-6.
11. Nelson, C.C., et al., The effects of P-box substitutions in thyroid hormone receptor on DNA binding specificity. Mol Endocrinol, 1994. 8(7): p. 829-40.
12. Rastinejad, F., et al., Structural determinants of nuclear receptor assembly on DNA direct repeats. Nature, 1995. 375(6528): p. 203-11.
13. Horlein, A.J., et al., Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature, 1995. 377(6548): p. 397-404.
14. Wagner, R.L., et al., A structural role for hormone in the thyroid hormone receptor. Nature, 1995. 378(6558): p. 690-7.
15. Baniahmad, A., et al., Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site. Cell, 1990. 61(3): p. 505-14.
16. Barra, G.B., et al., [Molecular mechanism of thyroid hormone action]. Arq Bras Endocrinol Metabol, 2004. 48(1): p. 25-39.
17. Naar, A.M., et al., The orientation and spacing of core DNA-binding motifs dictate selective transcriptional responses to three nuclear receptors. Cell, 1991. 65(7): p. 1267-79.
18. Harvey, C.B. and G.R. Williams, Mechanism of thyroid hormone action. Thyroid, 2002. 12(6): p. 441-6.
19. Zhang, X.K., et al., Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors. Nature, 1992. 355(6359): p. 441-6.
20. Polson, A.G., B.L. Bass, and J.L. Casey, RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase. Nature, 1996. 380(6573): p. 454-6.
21. Chakravarti, D., et al., Role of CBP/P300 in nuclear receptor signalling. Nature, 1996. 383(6595): p. 99-103.
22. Lazar, M.A., Thyroid hormone action: a binding contract. J Clin Invest, 2003. 112(4): p. 497-9.
23. Lin, K.H., et al., Expression of mutant thyroid hormone nuclear receptors in human hepatocellular carcinoma cells. Mol Carcinog, 1999. 26(1): p. 53-61.
24. Lin, K.H., et al., Identification of naturally occurring dominant negative mutants of thyroid hormone alpha 1 and beta 1 receptors in a human hepatocellular carcinoma cell line. Endocrinology, 1996. 137(10): p. 4073-81.
25. Kamiya, Y., et al., Expression of mutant thyroid hormone nuclear receptors is associated with human renal clear cell carcinoma. Carcinogenesis, 2002. 23(1): p. 25-33.
26. Li, Z., et al., Biallelic inactivation of the thyroid hormone receptor beta1 gene in early stage breast cancer. Cancer Res, 2002. 62(7): p. 1939-43.
27. Chen, R.N., et al., Thyroid hormone promotes cell invasion through activation of furin expression in human hepatoma cell lines. Endocrinology, 2008. 149(8): p. 3817-31.
28. Lee, R.C., R.L. Feinbaum, and V. Ambros, The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 1993. 75(5): p. 843-54.
29. Ruvkun, G., Molecular biology. Glimpses of a tiny RNA world. Science, 2001. 294(5543): p. 797-9.
30. Gregory, R.I. and R. Shiekhattar, MicroRNA biogenesis and cancer. Cancer Res, 2005. 65(9): p. 3509-12.
31. Calin, G.A., et al., Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A, 2004. 101(9): p. 2999-3004.
32. Volinia, S., et al., A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A, 2006. 103(7): p. 2257-61.
33. Zhang, Z., et al., miR-21 plays a pivotal role in gastric cancer pathogenesis and progression. Lab Invest, 2008. 88(12): p. 1358-66.
34. Chan, J.A., A.M. Krichevsky, and K.S. Kosik, MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res, 2005. 65(14): p. 6029-33.
35. Meng, F., et al., MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology, 2007. 133(2): p. 647-58.
36. Asangani, I.A., et al., MicroRNA-21 (miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer. Oncogene, 2008. 27(15): p. 2128-36.
37. Zhu, S., et al., MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res, 2008. 18(3): p. 350-9.
38. Frankel, L.B., et al., Programmed cell death 4 (PDCD4) is an important functional target of the microRNA miR-21 in breast cancer cells. J Biol Chem, 2008. 283(2): p. 1026-33.
39. Si, M.L., et al., miR-21-mediated tumor growth. Oncogene, 2007. 26(19): p. 2799-803.
40. Zhu, S., et al., MicroRNA-21 targets the tumor suppressor gene tropomyosin 1 (TPM1). J Biol Chem, 2007. 282(19): p. 14328-36.
41. Sayed, D., et al., MicroRNA-21 targets Sprouty2 and promotes cellular outgrowths. Mol Biol Cell, 2008. 19(8): p. 3272-82.
42. Papagiannakopoulos, T., A. Shapiro, and K.S. Kosik, MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells. Cancer Res, 2008. 68(19): p. 8164-72.
43. Loffler, D., et al., Interleukin-6 dependent survival of multiple myeloma cells involves the Stat3-mediated induction of microRNA-21 through a highly conserved enhancer. Blood, 2007. 110(4): p. 1330-3.
44. Fujita, S., et al., miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism. J Mol Biol, 2008. 378(3): p. 492-504.
45. Mertens, A.E., R.C. Roovers, and J.G. Collard, Regulation of Tiam1-Rac signalling. FEBS Lett, 2003. 546(1): p. 11-6.
46. Minard, M.E., et al., The role of the guanine nucleotide exchange factor Tiam1 in cellular migration, invasion, adhesion and tumor progression. Breast Cancer Res Treat, 2004. 84(1): p. 21-32.
47. Hordijk, P.L., et al., Inhibition of invasion of epithelial cells by Tiam1-Rac signaling. Science, 1997. 278(5342): p. 1464-6.
48. Morikawa, K., et al., Influence of organ environment on the growth, selection, and metastasis of human colon carcinoma cells in nude mice. Cancer Res, 1988. 48(23): p. 6863-71.
49. Bourguignon, L.Y., et al., Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration. J Cell Biol, 2000. 150(1): p. 177-91.
50. Fujita, S., et al., miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism. J Mol Biol, 2008. 378(3): p. 492-504.
51. Lim, L.P., et al., Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature, 2005. 433(7027): p. 769-73.
52. Ding, Y., et al., Overexpression of Tiam1 in hepatocellular carcinomas predicts poor prognosis of HCC patients. Int J Cancer, 2009. 124(3): p. 653-8.
53. Hiyoshi, Y., et al., MicroRNA-21 regulates the proliferation and invasion in esophageal squamous cell carcinoma. Clin Cancer Res, 2009. 15(6): p. 1915-22
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