簡易檢索 / 詳目顯示

回結果列表
研究生： 賴冠榮
Lai, Guan-Rong
論文名稱： 探討前列腺癌產生維生素D抗性的表觀遺傳機制
Epigenetic Mechanisms of Vitamin D Resistance in Prostate Cancer
指導教授： 丁慧如
Ting, Huei-Ju
顏賢章
Yan, Shian-Jang
學位類別： 碩士
Master
系所名稱： 醫學院 - 生理學研究所
Department of Physiology
論文出版年： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 69
中文關鍵詞： 前列腺癌 、維生素D 、DNA甲基化
外文關鍵詞： prostate cancer, vitamin D, DNA methylation
相關次數： 點閱：64  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 前列腺癌(prostate cancer)是目前全球男性癌症死亡率排名第二之癌症，雖然許多文獻均指出維生素D的活性形式－1,25-二羥維生素D3(1,25-dihydroxyvitamin D3)具有抑制前列腺癌增生、誘導凋亡等等抗癌效果，然而卻發現長期治療會造成抗藥性的產生，其發生機制尚未完全明瞭。過去文獻已闡明將1,25-二羥維生素D3去活性代謝的酵素CYP24A1因DNA甲基化降低而過度表達，而CYP24A1的過度表達可導致前列腺癌細胞對維生素D抗癌效果的反應減少，顯示DNA甲基化在產生維生素D抗性中扮演重要角色。透過篩選與DNA甲基化相關的酵素，我們發現DNA甲基轉移酶1以及3B (DNMT1 and 3B)的表現量與活性受到1,25-二羥維生素D3抑制。軟體預測在DNMT1以及3B的啟動子區域中含有大量推定的維生素D響應元件，顯示其可能受到維生素D的調控。我們透過冷光素酶報導基因測試，發現1,25-二羥維生素D3抑制DNMT3B啟動子序列的轉錄活性。除此之外，生物資訊分析指出在DNMT1以及3B的3′非轉譯區含有許多軟體預測的微小RNA(miRNA)結合位點，且文獻指出這些微小RNA受到1,25-二羥維生素D3上調，顯示1,25-二羥維生素D3可能透過調控微小RNA間接抑制DNMT1以及3B。透過即時定量聚合酶鏈鎖反應分析，發現這些微小RNA確實受到1,25-二羥維生素D3上調。並且由冷光素酶報導基因測試，揭示1,25-二羥維生素D3調控抑制含DNMT3B的3′非轉譯區的冷光素酶表現，顯示1,25-二羥維生素D3透過上調微小RNA進而抑制DNMTs。更進一步探討1,25-二羥維生素D3長期處理所導致DNA甲基化下降是否使維生素D抗性基因上調，我們透過DNA甲基化微陣列分析85萬個CpG位點的甲基化狀態，發現許多候選基因在具有維生素D抗性的前列腺細胞中，DNA甲基化減少且過度表達，這些候選基因可能在前列腺癌產生維生素D抗性中扮演重要角色。綜上所述，我們研究結果揭示1,25-二羥維生素D3透過降低啟動子轉錄活性與上調微小RNA，進而抑制DNMT3B，最終可能造成前列腺癌發展維生素D抗性。

    Prostate cancer (PCa) is one of the most common cancers leading to death in men. De-spite of anti-proliferation effects of 1α,25-dihydroxyvitamin D3 (1,25-VD) on PCa, PCa ul-timately develops resistance after a long-term 1,25-VD treatment. DNA demethyla-tion/hypomethylation of CYP24A1, encoding a key enzyme in 1,25 VD degradation, leads to CYP24A1 overexpression which has been linked to 1,25 VD resistance. However, the un-derlying mechanisms by which 1,25-VD induces DNA de-/hypo-methylation and eventually leads to 1,25-VD resistance in PCa, remains unclear. Here, by screening for enzymes in-volved in DNA methylation, we found that 1,25 VD reduces the expression levels and activ-ities of DNA methyltransferase 1 and 3B (DNMT1 and 3B). Software analysis revealed several putative vitamin D receptor (VDR) response elements (VDREs) in the promoter re-gions of both DNMT1 and 3B. By performing luciferase reporter assays, we demonstrated that 1,25-VD/VDR down-regulated DNMT3B promoter transactivity. Moreover, bioinfor-matics analysis predicted that several 1,25-VD-upregulated miRNAs could targets the 3′-UTR regions of DNMT1 and 3B. We first confirmed the 1,25-VD-induced upregulation of these miRNAs by qPCR; then the luciferase reporter assay revealed that 1,25-VD inhib-ited DNMT3B through regulating its 3′-UTR. Finally, DNA methylation status over 850,000 CpG sites were analyzed by methylation arrays in parental, vehicle long-term treated, and 1,25-VD long-term treated PCa cells. Several candidate genes were found to be hypometh-ylated and overexpressed in vitamin D resistant PCa cells. Taken together, our study reveals that 1,25-VD inhibit DNMT3B through suppressing promoter transactivity and 3′-UTR reg-ulation, which may lead to overexpression of genes resulting in vitamin D resistance in PCa.

    中文摘要………………………………………………………………………………I Abstract………………………………………………………………………………II Acknowledgements…………………………………………………………………III Index…………………………………………………………………………………IV Abbreviations……………………………………………………………………….VI 1. Introduction 1.1 Current issues in prostate cancer therapy…………………………………...1 1.2 Inhibitory effects of vitamin D on prostate cancer………………………….1 1.3 Vitamin D action……………………………………………………………...2 1.4 Loss of vitamin D responsiveness in prostate cancer……………………….2 1.5 DNA methylation in prostate cancer………………………………………...3 1.6 The role of DNA methylation in vitamin D responsiveness…………………4 1.7 Regulation of vitamin D on DNA methylation………………………………5 1.8 Regulation of vitamin D on miRNA………………………………………….5 2. Materials and methods 2.1 Cell culture……………………………………………………………………7 2.2 Plasmids and reagents………………………………………………………..7 2.3 Quantitative PCR (qPCR)……………………………………………………7 2.4 Bisulfite conversion…………………………………………………………...8 2.5 Methylation specific PCR (MS-PCR)………………………………………..8 2.6 Bisulfite sequencing…………………………………………………………..8 2.7 Methylation array…………………………………………………………….9 2.8 Protein isolation………………………………………………………………9 2.9 Western blotting……………………………………………………………10 2.10 DNMT activity assay……………………………………………………...10 2.11 Cell viability assay…..……………………………………………………10 2.12 Luciferase reporter assay………………………………………………...11 2.13 Gene ontology enrichment analysis……………………………………...11 3. Results 3.1 PCa cells develop resistance to the anti-proliferative effects of 1,25-VD upon long-term treatment with 1,25-VD……………..…………………….12 3.2 1,25-VD reduces DNMT3B through inhibition of promoter transactivity and 3′-UTR regulation………………………………………………………12 3.3 Long-term 1,25-VD treatment does not induce hypomethylation of NCoR1………………………………………………………………………..14 3.4 Long-term 1,25-VD treatment induces DNA hypomethylation of specific genes involved in the mTOR pathway……………………………………...14 3.5 Long-term 1,25-VD treatment induces DNA hypomethylation and overexpres-sion of specific genes involved in PCa progression……………………15 4. Discussion………………………………………………………………………..17 5. References……………………………………………………………………….21 6. Tables Table 1. List of primer sequences……………………………………………….25 Table 2. List of candidate miRNAs……………………………………………..27 Table 3. List of biological process enrichment of hypomethylated genes in vitamin D resistant PCa cells……………………………………………………28 Table 4. List of candidate genes………………………………………………...55 7. Figures Figure 1. 1,25-VD long-term treatment leads to development of vitamin D re-sistance in PCa cells…………………………………………………………..56 Figure 2. 1,25-VD reduces expressions and activities of DNMT1 and DNMT3B in PCa cells………………………………………………………………………58 Figure 3. 1,25-VD inhibit DNMT3B promoter transactivity in PCa cells……60 Figure 4. 1,25-VD inhibit DNMT3B through 3′-UTR regulation in PCa cells..61 Figure 5. 1,25-VD long-term treatment does not lead to altered methylation status near the transcription start site of NCoR1 in PCa cells………………..63 Figure 6. Specific genes are hypomethylated and overexpressed in vitamin D re-sistant PCa cells……………………………………………………………….65 Figure 7. Hypomethylated candidate gene expressions are correlated with PCa tumor grade……………………………………………………………………67 Figure 8. The role of 1,25-VD long-term treatment induced hypomethylated genes in PI3K/Akt, MAPK, Wnt and mTOR signaling pathways…………….68 Figure 9. The proposed model of long-term 1,25-VD treatment induced vitamin D resistance……………………………………………………………….……………..69

    1. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2018. CA Cancer J Clin, 2018. 68(1): p. 7-30.
    2. Banerjee, P.P., et al., Androgen action in prostate function and disease. Am J Clin Exp Urol, 2018. 6(2): p. 62-77.
    3. Krishnan, A.V., D.M. Peehl, and D. Feldman, Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression. J Cell Biochem, 2003. 88(2): p. 363-71.
    4. Gupta, D., et al., Vitamin D and prostate cancer risk: a review of the epidemiological literature. Prostate Cancer Prostatic Dis, 2009. 12: p. 215.
    5. Blutt, S.E., et al., Calcitriol-induced apoptosis in LNCaP cells is blocked by overexpression of Bcl-2. Endocrinology, 2000. 141(1): p. 10-7.
    6. Swami, S., A.V. Krishnan, and D. Feldman, Vitamin D metabolism and action in the prostate: implications for health and disease. Mol Cell Endocrinol, 2011. 347(1-2): p. 61-9.
    7. Yang, E.S. and K.L. Burnstein, Vitamin D inhibits G1 to S progression in LNCaP prostate cancer cells through p27Kip1 stabilization and Cdk2 mislocalization to the cytoplasm. J Biol Chem, 2003. 278(47): p. 46862-8.
    8. Bao, B.Y., et al., Down-regulation of NF-kappaB signals is involved in loss of 1alpha,25-dihydroxyvitamin D3 responsiveness. J Steroid Biochem Mol Biol, 2010. 120(1): p. 11-21.
    9. Huggins, C. and C.V. Hodges, Studies on Prostatic Cancer. I. The Effect of Castration, of Estrogen and of Androgen Injection on Serum Phosphatases in Metastatic Carcinoma of the Prostate. Cancer Res, 1941. 1(4): p. 293-297.
    10. Asmane, I., et al., New strategies for medical management of castration-resistant prostate cancer. Oncology, 2011. 80(1-2): p. 1-11.
    11. Schwartz, G.G. and B.S. Hulka, Is vitamin D deficiency a risk factor for prostate cancer? (Hypothesis). Anticancer Res, 1990. 10(5a): p. 1307-11.
    12. Miller, G.J., et al., The human prostatic carcinoma cell line LNCaP expresses biologically active, specific receptors for 1 alpha,25-dihydroxyvitamin D3. Cancer Res, 1992. 52(3): p. 515-20.
    13. Peehl, D.M., et al., Antiproliferative effects of 1,25-dihydroxyvitamin D3 on primary cultures of human prostatic cells. Cancer Res, 1994. 54(3): p. 805-10.
    14. Skowronski, R.J., D.M. Peehl, and D. Feldman, Vitamin D and prostate cancer: 1,25 dihydroxyvitamin D3 receptors and actions in human prostate cancer cell lines. Endocrinology, 1993. 132(5): p. 1952-60.
    15. Haussler, M.R., et al., Molecular mechanisms of vitamin D action. Calcif Tissue Int, 2013. 92(2): p. 77-98.
    16. Pike, J.W. and S. Christakos, Biology and Mechanisms of Action of the Vitamin D Hormone. Endocrinol Metab Clin North Am, 2017. 46(4): p. 815-843.
    17. Ting, H.J., et al., Increased expression of corepressors in aggressive androgen-independent prostate cancer cells results in loss of 1alpha,25-dihydroxyvitamin D3 responsiveness. Mol Cancer Res, 2007. 5(9): p. 967-80.
    18. Pálmer, H.G., et al., The transcription factor SNAIL represses vitamin D receptor expression and responsiveness in human colon cancer. Nat Med, 2004. 10: p. 917.
    19. Zhuang, S.H., et al., Vitamin D receptor content and transcriptional activity do not fully predict antiproliferative effects of vitamin D in human prostate cancer cell lines. Mol Cell Endocrinol, 1997. 126(1): p. 83-90.
    20. Khanim, F.L., et al., Altered SMRT levels disrupt vitamin D3 receptor signalling in prostate cancer cells. Oncogene, 2004. 23(40): p. 6712-25.
    21. Tannour-Louet, M., et al., Increased expression of CYP24A1 correlates with advanced stages of prostate cancer and can cause resistance to vitamin D3-based therapies. FASEB J, 2014. 28(1): p. 364-72.
    22. Muindi, J.R., et al., CYP24A1 inhibition enhances the antitumor activity of calcitriol. Endocrinology, 2010. 151(9): p. 4301-12.
    23. Ly, L.H., et al., Liarozole Acts Synergistically with 1α,25-Dihydroxyvitamin D3 to Inhibit Growth of DU 145 Human Prostate Cancer Cells by Blocking 24-Hydroxylase Activity*. Endocrinology, 1999. 140(5): p. 2071-2076.
    24. Peehl, D.M., et al., Preclinical activity of ketoconazole in combination with calcitriol or the vitamin D analogue EB 1089 in prostate cancer cells. J Urol, 2002. 168(4 Pt 1): p. 1583-8.
    25. Fetahu, I.S., J. Hobaus, and E. Kallay, Vitamin D and the epigenome. Front Physiol, 2014. 5: p. 164.
    26. Zelic, R., et al., Global DNA hypomethylation in prostate cancer development and progression: a systematic review. Prostate Cancer Prostatic Dis, 2015. 18(1): p. 1-12.
    27. Kohli, R.M. and Y. Zhang, TET enzymes, TDG and the dynamics of DNA demethylation. Nature, 2013. 502(7472): p. 472-9.
    28. Canova, C., et al., Epigenetic control of programmed cell death: inhibition by 5-azacytidine of 1,25-dihydroxyvitamin D3-induced programmed cell death in C6.9 glioma cells. Mech Ageing Dev, 1998. 101(1-2): p. 153-66.
    29. Marik, R., et al., DNA methylation-related vitamin D receptor insensitivity in breast cancer. Cancer Biol Ther, 2010. 10(1): p. 44-53.
    30. Luo, W., et al., Epigenetic regulation of vitamin D 24-hydroxylase/CYP24A1 in human prostate cancer. Cancer Res, 2010. 70(14): p. 5953-62.
    31. Tapp, H.S., et al., Nutritional factors and gender influence age-related DNA methylation in the human rectal mucosa. Aging Cell, 2013. 12(1): p. 148-55.
    32. Rawson, J.B., et al., Vitamin D intake is negatively associated with promoter methylation of the Wnt antagonist gene DKK1 in a large group of colorectal cancer patients. Nutr Cancer, 2012. 64(7): p. 919-28.
    33. Aguilera, O., et al., The Wnt antagonist DICKKOPF-1 gene is induced by 1α,25-dihydroxyvitamin D 3 associated to the differentiation of human colon cancer cells. Carcinogenesis, 2007. 28(9): p. 1877-1884.
    34. Pendas-Franco, N., et al., Vitamin D and Wnt/beta-catenin pathway in colon cancer: role and regulation of DICKKOPF genes. Anticancer Res, 2008. 28(5a): p. 2613-23.
    35. Lopes, N., et al., 1Alpha,25-dihydroxyvitamin D3 induces de novo E-cadherin expression in triple-negative breast cancer cells by CDH1-promoter demethylation. Anticancer Res, 2012. 32(1): p. 249-57.
    36. Doig, C.L., Recruitment of NCOR1 to VDR target genes is enhanced in prostate cancer cells and associates with altered DNA methylation patterns. Carcinogenesis, 2013. 34(2): p. 248-56.
    37. Jiang, F., et al., G2/M arrest by 1,25-dihydroxyvitamin D3 in ovarian cancer cells mediated through the induction of GADD45 via an exonic enhancer. J Biol Chem, 2003. 278(48): p. 48030-40.
    38. Zhang, X., S.V. Nicosia, and W. Bai, Vitamin D receptor is a novel drug target for ovarian cancer treatment. Curr Cancer Drug Targets, 2006. 6(3): p. 229-44.
    39. Bremmer, F., et al., Expression and function of the vitamin D receptor in malignant germ cell tumour of the testis. Anticancer Res, 2012. 32(1): p. 341-9.
    40. Niehrs, C. and A. Schafer, Active DNA demethylation by Gadd45 and DNA repair. Trends Cell Biol, 2012. 22(4): p. 220-7.
    41. Zeljic, K., G. Supic, and Z. Magic, New insights into vitamin D anticancer properties: focus on miRNA modulation. Mol Genet Genomics, 2017. 292(3): p. 511-524.
    42. Wang, W.L., et al., Effects of 1alpha,25 dihydroxyvitamin D3 and testosterone on miRNA and mRNA expression in LNCaP cells. Mol Cancer, 2011. 10: p. 58.
    43. Ting, H.J., et al., Identification of microRNA-98 as a therapeutic target inhibiting prostate cancer growth and a biomarker induced by vitamin D. J Biol Chem, 2013. 288(1): p. 1-9.
    44. Li, L.C. and R. Dahiya, MethPrimer: designing primers for methylation PCRs. Bioinformatics, 2002. 18(11): p. 1427-31.
    45. Messeguer, X., et al., PROMO: detection of known transcription regulatory elements using species-tailored searches. Bioinformatics, 2002. 18(2): p. 333-4.
    46. Fabbri, M., et al., MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc Natl Acad Sci U S A, 2007. 104(40): p. 15805-10.
    47. Chou, C.H., et al., miRTarBase update 2018: a resource for experimentally validated microRNA-target interactions. Nucleic Acids Res, 2018. 46(D1): p. D296-d302.
    48. Nguyen, T.P., A.R. Frank, and J.L. Jewell, Amino acid and small GTPase regulation of mTORC1. Cell Logist, 2017. 7(4): p. e1378794.
    49. Houles, T. and P.P. Roux, Defining the role of the RSK isoforms in cancer. Semin Cancer Biol, 2018. 48: p. 53-61.
    50. Meng, D., A.R. Frank, and J.L. Jewell, mTOR signaling in stem and progenitor cells. Development, 2018. 145(1).
    51. Dienstmann, R., et al., Picking the point of inhibition: a comparative review of PI3K/AKT/mTOR pathway inhibitors. Mol Cancer Ther, 2014. 13(5): p. 1021-31.
    52. Kuck, D., et al., Nanaomycin A selectively inhibits DNMT3B and reactivates silenced tumor suppressor genes in human cancer cells. Mol Cancer Ther, 2010. 9(11): p. 3015-23.
    53. Zagorac, S., et al., DNMT1 Inhibition Reprograms Pancreatic Cancer Stem Cells via Upregulation of the miR-17-92 Cluster. Cancer Res, 2016. 76(15): p. 4546-4558.
    54. Gailhouste, L., et al., Epigenetic reprogramming using 5-azacytidine promotes an anti-cancer response in pancreatic adenocarcinoma cells. Cell Death Dis, 2018. 9(5).
    55. Rajabi, H., et al., DNA METHYLATION BY DNMT1 AND DNMT3b METHYLTRANSFERASES IS DRIVEN BY THE MUC1-C ONCOPROTEIN IN HUMAN CARCINOMA CELLS. Oncogene, 2016. 35(50): p. 6439-6445.
    56. Denis, H., M.N. Ndlovu, and F. Fuks, Regulation of mammalian DNA methyltransferases: a route to new mechanisms. EMBO Rep, 2011. 12(7): p. 647-56.
    57. Lopez de Silanes, I., et al., The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA. Nucleic Acids Res, 2009. 37(8): p. 2658-71.
    58. Narayanan, R., et al., MicroRNAs Are Mediators of Androgen Action in Prostate and Muscle. PLoS ONE, 2010. 5(10): p. e13637.
    59. Zhang, B., et al., Upregulated microRNA-199a-5p inhibits nuclear receptor corepressor 1 translation in mice with nonalcoholic steatohepatitis. Mol Med Rep, 2014. 10(6): p. 3080-6.
    60. Alimirah, F., et al., Crosstalk between the vitamin D receptor (VDR) and miR-214 in regulating SuFu, a hedgehog pathway inhibitor in breast cancer cells. Exp Cell Res, 2016. 349(1): p. 15-22.
    61. Bao, B.-Y., et al., Androgen signaling is required for the vitamin D-mediated growth inhibition in human prostate cancer cells. Oncogene, 2004. 23: p. 3350.
    62. Pópulo, H., J.M. Lopes, and P. Soares, The mTOR Signalling Pathway in Human Cancer. Int J Mol Sci, 2012. 13(2): p. 1886-1918.
    63. Liu, E., et al., Control of mTORC1 signaling by the Opitz syndrome protein MID1. Proc Natl Acad Sci U S A, 2011. 108(21): p. 8680-5.
    64. Wlodarchak, N. and Y. Xing, PP2A as a master regulator of the cell cycle. Crit Rev Biochem Mol Biol, 2016. 51(3): p. 162-184.
    65. Garcia-Marcos, M., P. Ghosh, and M.G. Farquhar, GIV/Girdin Transmits Signals from Multiple Receptors by Triggering Trimeric G Protein Activation. J Biol Chem, 2015. 290(11): p. 6697-6704.
    66. Guo, L.S., et al., Synergistic antitumor activity of vitamin D3 combined with metformin in human breast carcinoma MDA-MB-231 cells involves m-TOR related signaling pathways. Pharmazie, 2015. 70(2): p. 117-22.
    67. Li, H.X., et al., Vitamin D3 potentiates the growth inhibitory effects of metformin in DU145 human prostate cancer cells mediated by AMPK/mTOR signalling pathway. Clin Exp Pharmacol Physiol, 2015. 42(6): p. 711-7.
    68. Kanehisa, M. and S. Goto, KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res, 2000. 28(1): p. 27-30.
    69. Henderson, D.J.P., et al., The cAMP phosphodiesterase-4D7 (PDE4D7) is downregulated in androgen-independent prostate cancer cells and mediates proliferation by compartmentalising cAMP at the plasma membrane of VCaP prostate cancer cells. Br J Cancer, 2014. 110(5): p. 1278-1287.
    70. Reams, R.R., et al., Immunohistological Analysis of ABCD3 Expression in Caucasian and African American Prostate Tumors. Biomed Res Int, 2015. 2015: p. 132981.
    71. Murillo-Garzon, V. and R. Kypta, WNT signalling in prostate cancer. Nat Rev Urol, 2017. 14(11): p. 683-696.
    72. Yue, W., et al., Downregulation of Dkk3 activates beta-catenin/TCF-4 signaling in lung cancer. Carcinogenesis, 2008. 29(1): p. 84-92.
    73. Yang, Y., L. Quan, and Y. Ling, RBMS3 Inhibits the Proliferation and Metastasis of Breast Cancer Cells. Oncol Res, 2018. 26(1): p. 9-15.
    74. Larriba, M.J., et al., Vitamin D Is a Multilevel Repressor of Wnt/β-Catenin Signaling in Cancer Cells. Cancers, 2013. 5(4): p. 1242-1260.

    無法下載圖示 校內：2023-07-13公開
    校外：不公開
    電子論文尚未授權公開，紙本請查館藏目錄
    QR CODE