臺灣博碩士論文加值系統

microRNA為一個內源性單股的小RNA分子，且miRNA參與了基因的調控。miRNAs在不同的物種中均已被確認其存在性，像是果蠅、老鼠和人類等等。miRNA的基因可以位於introns或是exons中，也可能位於基因間的區段中，並且miRNA是由RNA聚合酶II或是RNA聚合酶III所轉錄出來的。miRNAs為非常重要的基因調控者，其參與了細胞生長、細胞增生、細胞分化和細胞死亡的機制。在最近的研究中已經發現了miRNAs突變、大量增加、miRNAs缺失或是epigenetic silencing都與許多的人類疾病和癌症有相關。因此 miRNAs也許有tumor suppressor或是oncogene的功能。Epithelial-mesenchymal transition為癌症轉移的一個重要的過程，EMT會去增加癌症細胞遷移和侵入的能力。我們的研究主要在於尋找是否有miRNAs參與了受到hypoxia誘發的epithelial-mesenchymal transition，並了解miRNA在其中所扮演的角色。於是我們利用了兩個可以預測會影響HDAC3的miRNAS的程式: miRBASE、TargetScan，並分析出了miR-X會去影響EMT相關的兩個蛋白質: HDAC3和SENP1。我的初步研究觀察到了miR-X可以去抑制HDAC3和SENP1的luciferse activity，並且在stable clone中也觀察到了HDAC3和SENP1的蛋白質有被抑制的情形。同時我們也觀察到了miR-X可以去抑制癌症細胞的遷移和侵入的能力。根據上述的實驗結果，我們推斷miR-X可以對癌症轉移有抑制的可能性。但是是什麼去調控miR-X仍然是未知的，所以我們接下來會朝著hypoxia是如何去調控miR-X的表現和miR-X是如何去抑制HDAC3和SENP1做為未來的研究方向，並去檢測乳癌細胞中的miR-X的表現量去測試其癒後的價值來預測對病人的存活率的影響。

MicroRNAs are endogenous, single-strand RNA molecules that regulate gene expression. MicroRNAs have been identified in different species such as Drosophila, mouse and human. MicroRNA genes located within introns, exons or intergenic areas were transcribed by RNA polymerase II or RNA polymerase III. MicroRNAs are important gene regulators that control cell growth, cell proliferation, cell differentiation and cell death. Recent results have shown that microRNA mutation, amplification, deletion or epigenetic silencing was correlated with various human diseases and cancers. Therefore, microRNA may function as tumor suppressor genes or oncogenes. Epithelial-mesenchymal transition is an important process in cancer metastasis which promote cell migration and cell invasion in cancers. We were interested in identifying novel microRNA(s) involved in epithelial-mesenchymal transition that were induced by hypoxia. From two different predict programs miRBase, TargetScan, the analysis showed that miR-X could target to EMT-relative gene HDAC3 and SENP1. Our preliminary data showed that miR-X could repress luciferase activity of 3’UTR of HDAC3 and SENP1, and the expression of HDAC3 and SENP1 were also repressed in miR-X overexpressing stable lines. miR-X also repress cancer cells migration and invasion ability. For this proposal, I will investigate how hypoxia regulates miR-X expression, and determine how miR-X inhibits the expression of HDAC3 and SENP1. The expression level of miR-X will also be conflated with breast cancer samples to test its prognostic value to predict patients’ survival.

Contents
中文摘要 - 1 -
Abstract - 2 -
1. Introduction - 3 -
A. MicroRNA biogenesis - 3 -
B. Cancer metastasis and epithelial-to-mesenchymal transition - 5 -
C. Epithelial-to-mesenchymal transition - 5 -
D. Hypoxia - 6 -
E. Hypoxia-inducible factor-1 (HIF-1) - 8 -
F. Histone deacetylase 3 - 9 -
G. SUMO-specific protease 1 - 11 -
2. Result - 13 -
miR-X can repress HDAC3 at the mRNA and protein levels. - 13 -
miR-X can repress SENP1 and HDAC3 at the mRNA and protein levels in the H1299 cancer cell line. - 14 -
miR-X expression level is down-regulated in hypoxia - 15 -
miR-X can repress SENP1 and HDAC3 at the mRNA and protein levels in the MCF7 cancer cell line. - 16 -
Overexrpession of miR-X inhibited the invasion and migration of MCF7 cells. - 17 -
3. Discussion - 18 -
4. Experimental materials and methods - 19 -
5. Figures - 24 -
Figure1. miRNAs computationally predicted to target HDAC3. - 24 -
Figure2. Luciferase activity of HDAC3 3’UTR is regulated by microRNAs. - 25 -
Figure3. Stable express the miRNAs in MCF7 cell line. - 26 -
Figure4. Protein expression of HDAC3 was repressed by miR-X. - 27 -
Figure5. The effect of miR-X can be removed by mutating HDAC3 3’UTR-miR-X binding site. - 28 -
Figure6. miR-X predicted target genes. - 29 -
Figure7. The effect of miR-X on luciferase activity of reporter vectors with wild-type or mutant SENP1 3’UTR. - 30 -
Figure8. Stable express the miRNAs in H1299 cell line. - 31 -
Figure9. Protein expression of HDAC3 and SENP1 were repressed by miR-X. - 32 -
Figure10. miR-X expression level is down-regulated under hypoxia. - 33 -
Figure11. Stable express the miRNAs in MCF7 cell line. - 34 -
Figure12. Protein expression of HDAC3 and SENP1 were repressed by miR-X. - 35 -
Figure13. Repression of HDAC3 and SENP1 mRNA levels can be rescued by miR-X inhibitor. - 36 -
Figure14. Repression of HDAC3 and SENP1 protein levels can be rescued by miR-X inhibitor. - 37 -
Figure15. miR-X can suppress migration activity. - 38 -
Figure16. miR-X can suppress invasion activity. - 39 -
Figure17. The working model. - 40 -
6. Table - 41 -
Table 1. List of proteins tested by and characteristics of the corresponding antibodies - 41 -
Table 2. Sequence of the oligonuleotides for constructions - 41 -
Table 3. Plasmid construction - 42 -
7. Reference - 43 -

1. Ahringer, J. (2000). "NuRD and SIN3 histone deacetylase complexes in development." Trends Genet 16(8): 351-356.

2. Annicotte, J. S., I. Iankova, et al. (2006). "Peroxisome proliferator-activated receptor gamma regulates E-cadherin expression and inhibits growth and invasion of prostate cancer." Mol Cell Biol 26(20): 7561-7574.

3. Bailey, D. and P. O'Hare (2004). "Characterization of the localization and proteolytic activity of the SUMO-specific protease, SENP1." J Biol Chem 279(1): 692-703.

4. Bawa-Khalfe, T., J. Cheng, et al. (2007). "Induction of the SUMO-specific protease 1 transcription by the androgen receptor in prostate cancer cells." J Biol Chem 282(52): 37341-37349.

5. Bawa-Khalfe, T. and E. T. Yeh (2010). "The in vivo functions of desumoylating enzymes." Subcell Biochem 54: 170-183.

6. Bhaskara, S., B. J. Chyla, et al. (2008). "Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control." Mol Cell 30(1): 61-72.

7. Bhaskara, S., S. K. Knutson, et al. (2010). "Hdac3 is essential for the maintenance of chromatin structure and genome stability." Cancer Cell 18(5): 436-447.

8. Cheng, J., T. Bawa, et al. (2006). "Role of desumoylation in the development of prostate cancer." Neoplasia 8(8): 667-676.

9. Cheng, J., X. Kang, et al. (2007). "SUMO-specific protease 1 is essential for stabilization of HIF1alpha during hypoxia." Cell 131(3): 584-595.

10. Cheng, J., N. D. Perkins, et al. (2005). "Differential regulation of c-Jun-dependent transcription by SUMO-specific proteases." J Biol Chem 280(15): 14492-14498.

11. Cheng, J., D. Wang, et al. (2004). "SENP1 enhances androgen receptor-dependent transcription through desumoylation of histone deacetylase 1." Mol Cell Biol 24(13): 6021-6028.

12. Chou, C. W., M. S. Wu, et al. (2011). "HDAC inhibition decreases the expression of EGFR in colorectal cancer cells." PLoS One 6(3): e18087.
Escaffit, F., O. Vaute, et al. (2007). "Cleavage and cytoplasmic relocalization of histone deacetylase 3 are important for apoptosis progression." Mol Cell Biol 27(2): 554-567.

13. Esquela-Kerscher, A. and F. J. Slack (2006). "Oncomirs - microRNAs with a role in cancer." Nat Rev Cancer 6(4): 259-269.

14. Fajas, L., V. Egler, et al. (2002). "The retinoblastoma-histone deacetylase 3 complex inhibits PPARgamma and adipocyte differentiation." Dev Cell 3(6): 903-910.

15. Garzon, R., G. A. Calin, et al. (2009). "MicroRNAs in Cancer." Annu Rev Med 60: 167-179.

16. Gennarino, V. A., M. Sardiello, et al. (2009). "MicroRNA target prediction by expression analysis of host genes." Genome Res 19(3): 481-490.

17. Guenther, M. G., O. Barak, et al. (2001). "The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3." Mol Cell Biol 21(18): 6091-6101.

18. Hartman, H. B., J. Yu, et al. (2005). "The histone-binding code of nuclear receptor co-repressors matches the substrate specificity of histone deacetylase 3." EMBO Rep 6(5): 445-451.
Jepsen, K. and M. G. Rosenfeld (2002). "Biological roles and mechanistic actions of co-repressor complexes." J Cell Sci 115(Pt 4): 689-698.

19. Joglekar, M. V., D. Patil, et al. (2009). "The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells." Islets 1(2): 137-147.

20. Karagianni, P. and J. Wong (2007). "HDAC3: taking the SMRT-N-CoRrect road to repression." Oncogene 26(37): 5439-5449.

21. Kim, V. N. and J. W. Nam (2006). "Genomics of microRNA." Trends Genet 22(3): 165-173.

22. Kwon, H. J., M. S. Kim, et al. (2002). "Histone deacetylase inhibitor FK228 inhibits tumor angiogenesis." Int J Cancer 97(3): 290-296.
Lane, A. A. and B. A. Chabner (2009). "Histone deacetylase inhibitors in cancer therapy." J Clin Oncol 27(32): 5459-5468.

23. Li, J., S. Donath, et al. (2010). "miR-30 regulates mitochondrial fission through targeting p53 and the dynamin-related protein-1 pathway." PLoS Genet 6(1): e1000795.

24. Li, X., Y. Luo, et al. (2008). "SENP1 mediates TNF-induced desumoylation and cytoplasmic translocation of HIPK1 to enhance ASK1-dependent apoptosis." Cell Death Differ 15(4): 739-750.

25. Liu, J., M. Zheng, et al. (2011). "MicroRNAs, an active and versatile group in cancers." Int J Oral Sci 3(4): 165-175.

26. Lu, J., G. Getz, et al. (2005). "MicroRNA expression profiles classify human cancers." Nature 435(7043): 834-838.

27. McQuown, S. C. and M. A. Wood (2011). "HDAC3 and the molecular brake pad hypothesis." Neurobiol Learn Mem 96(1): 27-34.

28. Mukhopadhyay, D. and M. Dasso (2007). "Modification in reverse: the SUMO proteases." Trends Biochem Sci 32(6): 286-295.

29. Pandey, M., P. Kaur, et al. (2011). "Plant flavone apigenin inhibits HDAC and remodels chromatin to induce growth arrest and apoptosis in human prostate cancer cells: In vitro and in vivo study." Mol Carcinog.

30. Rajendran, P., B. Delage, et al. (2011). "Histone deacetylase turnover and recovery in sulforaphane-treated colon cancer cells: competing actions of 14-3-3 and Pin1 in HDAC3/SMRT corepressor complex dissociation/reassembly." Mol Cancer 10: 68.

31. Sayed, D. and M. Abdellatif (2011). "MicroRNAs in development and disease." Physiol Rev 91(3): 827-887.

32. Strahl, B. D. and C. D. Allis (2000). "The language of covalent histone modifications." Nature 403(6765): 41-45.

33. Ulrich, H. D. (2007). "SUMO teams up with ubiquitin to manage hypoxia." Cell 131(3): 446-447.

34. van Kouwenhove, M., M. Kedde, et al. (2011). "MicroRNA regulation by RNA-binding proteins and its implications for cancer." Nat Rev Cancer 11(9): 644-656.

35. Wu, L. M., Z. Yang, et al. (2010). "Identification of histone deacetylase 3 as a biomarker for tumor recurrence following liver transplantation in HBV-associated hepatocellular carcinoma." PLoS One 5(12): e14460.

36. Wu, M. Z., Y. P. Tsai, et al. (2011). "Interplay between HDAC3 and WDR5 is essential for hypoxia-induced epithelial-mesenchymal transition." Mol Cell 43(5): 811-822.

37. Xu, Y., Y. Zuo, et al. (2010). "Induction of SENP1 in endothelial cells contributes to hypoxia-driven VEGF expression and angiogenesis." J Biol Chem 285(47): 36682-36688.

38. Yang, J. S. and E. C. Lai (2011). "Alternative miRNA biogenesis pathways and the interpretation of core miRNA pathway mutants." Mol Cell 43(6): 892-903.

39. Yang, W. M., S. C. Tsai, et al. (2002). "Functional domains of histone deacetylase-3." J Biol Chem 277(11): 9447-9454.

40. Yang, W. M., Y. L. Yao, et al. (1997). "Isolation and characterization of cDNAs corresponding to an additional member of the human histone deacetylase gene family." J Biol Chem 272(44): 28001-28007.

41. Yang, Y., W. Fu, et al. (2007). "SIRT1 sumoylation regulates its deacetylase activity and cellular response to genotoxic stress." Nat Cell Biol 9(11): 1253-1262.

42. Yeh, E. T. (2009). "SUMOylation and De-SUMOylation: wrestling with life's processes." J Biol Chem 284(13): 8223-8227.

43. Ying, S. Y. and S. L. Lin (2005). "Intronic microRNAs." Biochem Biophys Res Commun 326(3): 515-520.

44. Yoon, H. G., Y. Choi, et al. (2005). "Reading and function of a histone code involved in targeting corepressor complexes for repression." Mol Cell Biol 25(1): 324-335.

45. Yu, F., H. Deng, et al. (2010). "Mir-30 reduction maintains self-renewal and inhibits apoptosis in breast tumor-initiating cells." Oncogene 29(29): 4194-4204.

46. Zhang, J., M. Kalkum, et al. (2002). "The N-CoR-HDAC3 nuclear receptor corepressor complex inhibits the JNK pathway through the integral subunit GPS2." Mol Cell 9(3): 611-623.