臺灣博碩士論文加值系統

MicroRNA-122(miR-122)是一在成熟肝臟中大量表現的微小RNA，在過去的研究中已知miR-122具有抑制肝癌生成及調節肝臟代謝的功能。有趣的是在Mir-122-/-小鼠的肝臟中，有較多會釋出發炎激素TNF-α及IL-6的巨噬細胞、嗜中性球等免疫細胞的浸潤，另外，在B型肝炎病人肝臟的miR-122表現量也較正常人表現的低，因此推測miR-122可能亦具有抗發炎之功能，而在我們先前的研究利用Mir-122-/-小鼠，也發現在KO小鼠的肝臟中，有較高的Ccl2、Ccl4、Ccl20、Cxcl2及Cxcl9之表現量，但是目前對於miR-122在人類肝臟中是否調控發炎仍不清楚。在本篇研究中，利用過量表現miR-122及抑制miR-122表現的肝癌細胞株模型，我們發現在以TNF-α刺激之後，miR-122在不同細胞株分別會調控CCL2、CCL5、CXCL8與CXCL10的mRNA表現，其中， CXCL10之表現在不同肝癌細胞株中都受到miR-122的調控。此外，在肝癌病人組織中，我們發現在肝癌相鄰非腫瘤組織中miR-122分別會與CCL2、CCL20及CXCL10之mRNA表現量呈現負相關，因此，我們進一步去探討miR-122調控趨化素表現的機制，結果發現，miR-122並不是透過抑制NF-κB的活性，來調控趨化素的表現，我們接著找到在CCL2及CXCL8的3’UTR上有miR-122的標靶位置，並經reporter assay實驗證明CCL2與CXCL8為miR-122之標靶基因。總結我們的實驗結果，發現在人類肝臟中，miR-122可能會藉由調控趨化素之表現，來抑制肝臟發炎的現象。

MicroRNA-122 (miR-122) is the most abundant microRNA in the adult liver. Previous studies have demonstrated the important function of miR-122 in the liver metabolism and suppression of tumor progression. In addition, there are evidence revealing an anti-inflammatory function of miR-122 in both humans and mice. In the Mir-122-/- mice, an increase in Kupffer cells and infiltrating leukocytes including monocytes and neutrophils which produce inflammatory cytokines IL-6 and TNF-α was detected in the liver. We also observed a higher expression of Ccl2、Ccl4、Ccl20、Cxcl2 and Cxcl10 in the -Mir-122-/- mice liver; however, whether miR-122 regulates inflammation in humans is still unclear. In this study, using overexpression and knockdown of miR-122 expression in human hepatoma cell lines, we found that TNF-α-induced chemokines, such as CCL2、CCL5、CXCL8 and CXCL10 were regulated by miR-122 in different cell lines. Among them, we consistently observed a suppression of CXCL10 expression by miR-122 in all cell lines. In the liver tissues from hepatocellular carcinoma patients, we found that in the adjacent non-tumor tissues, the expression of cytokine IL-6 and IL-22 as well as chemokine CCL5 and CXCL8 was higher than that in the tumor counterparts. Futhermore, miR-122 levels negatively correlated with CCL2、CCL20 and CXCL10 levels in the adjacent non-tumor tissue. These results indicate that miR-122 negatively regulate chemokine expression in humans. To further study the mechanism by which miR-122 regulates chemokine, we showed that the NF-κB activity was not changed by miR-122. Instead we identified CCL2 and CXCL8 as miR-122 target genes. Because there is no miR-122 target site in the CXCL10 transcript, we assume that miR-122 modulates its expression in a indirect manner. In conclusion, we found that in human liver, miR-122 may regulate the expression of chemokine to control liver inflammation.

目錄
目錄.............................i
圖表目錄........................ ii
中文摘要 ........................iv
Abstract.........................v
第一章　緒論......................1
1.1　MicroRNA-122................1
1.2　肝細胞癌.....................3
1.3　肝臟慢性發炎與肝癌............3
1.4　miR-122與肝臟發炎............4
1.5　趨化素與肝臟發炎、肝癌........4
1.6　研究動機.....................5
第二章　材料與方法.................7
2.1　材料.........................7
2.2　方法........................14
第三章 實驗結果...................23
第四章 討論.......................29
參考資料..........................33
圖表..............................39
附錄..............................61


圖表目錄
圖一、miR-122在人類肝臟細胞株的表現與建立過量表現miR-122之人類肝癌細胞株..........................................40
圖二、在 HepG2細胞中過量表現miR-122會降低TNF-α刺激後CCL2、CXCL8、CXCL10 mRNA表現量..............................41
圖三、TNF-α無法刺激Hep3B細胞株表現特定趨化素............42
圖四、在 SK-Hep-1細胞中過量表現miR-122會降低TNF-α刺激後CCL5 、CXCL8及CXCL10 mRNA表現量............................43
圖五、在 SK-Hep-1細胞中過量表現miR-122會降低TNF-α刺激後CXCL8蛋白表現量............................................44
圖六、在 Huh7細胞中抑制miR-122會使TNF-α刺激後CXCL10 mRNA表現量上升...............................................46
圖七、在 Huh7細胞中抑制miR-122會使TNF-α刺激後CXCL10 蛋白表現量上升，但不影響CXCL8蛋白表現量........................47
圖八、肝癌相鄰的非腫瘤組織與腫瘤組織之miR-122表現量並無顯著差異...................................................48
圖九、肝癌相鄰非腫瘤組織之CCL5與CXCL8 mRNA表現量較腫瘤組織高 ......................................................49
圖十、肝癌相鄰非腫瘤組織之IL-6與IL-22 mRNA表現量較腫瘤組織並無顯著差異.............................................50
圖十一、肝癌相鄰的非腫瘤組織與腫瘤組織之RORγt mRNA表現量並無顯著差異................................................51
圖十二、肝癌相鄰的非腫瘤組織中miR-122表現量與CCL2及CCL20之mRNA表現量有顯著的負相關性.............................52
圖十三、肝癌組織中miR-122表現量與CCL5 mRNA表現量有顯著的負相關性.................................................53
圖十四、腫瘤組織miR-122表現量低於非腫瘤組織之病人，其非腫瘤組織中miR-122個別與CCL2及CXCL10之mRNA表現量呈現負相關.....54
圖十五、在HepG2細胞中過量表現miR-122不影響TNF-α引發之NF-κB的活性.................................................55
圖十六、人類CCL2與CXCL8基因為miR-122之目標基因.........57
表一、24位肝癌病人之臨床病理分析表.....................58
表二、肝癌相鄰的非腫瘤組織與肝癌組織之miR-122與chemokine mRNA表現量(臨床病理分析結果如表一).....................59
表三、肝癌相鄰的非腫瘤組織與肝癌組織之miR-122與chemokine mRNA表現量...........................................60
附錄一、包裝過量表現miR-122之慢病毒的載體pLAS2w.Ppuro和pLAS2w.Ppuro-miR-122................................61
附錄二、包裝抑制miR-122表現之慢病毒的載體pmiRZip和pmiRZip-122.................................................62
附錄三、NF-κB reporter assay之pLKO.AS3W NF-κB RE LUC 2P.puro載體.........................................63
附錄四、pRL-TK載體...................................64
附錄五、3’UTR reporter assay之表現螢光酵素psi-CHECK2載體 65
附錄六、5’UTR reporter assay之表現螢光酵素pMIR-REPORT載體 66

1. Girard, M., E. Jacquemin, A. Munnich, S. Lyonnet, A. Henrion-Caude. 2008. miR-122, a paradigm for the role of microRNAs in the liver. J Hepatol 48:648–656.
2. Xu, H., J. H. He, Z. D. Xiao, Q. Q. Zhang, Y. Q. Chen, H. Zhou, and L. H. Qu. 2010. Liver-enriched transcription factors regulate microRNA-122 that targets CUTL1 during liver development. Hepatology 52:1431-1442
3. Laudadio, I., I. Manfroid, Y. Achouri, D. Schmidt, M. D. Wilson, S. Cordi, L. Thorrez, L. Knoops, P. Jacquemin, F. Schuit, C. E. Pierreux, D. T. Odom, B. Peers, and F. P. Lemaigre. 2012. A feedback loop between the liver-enriched transcription factor network and miR-122 controls hepatocyte differentiation. Gastroenterology 142: 119-129.
4. Esau, C., S. Davis, S. F. Murray, X. X. Yu, S. K. Pandey, M. Pear, L. Watts, S. L. Booten, M. Graham, R. McKay, A. Subramaniam, S. Propp, B. A. Lollo, S. Freier, C. F. Bennett, S. Bhanot, and B. P. Monia. 2006. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab 3: 87-98.
5. Chen, Y., A. Shen, P. J. Rider, Y. Yu, K. Wu, Y. Mu, Q. Hao, Y. Liu, H. Gong, Y. Zhu, F. Liu, and J. Wu. 2011. A liver-specific microRNA binds to a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication. FASEB J 25: 4511-4521.
6. Wang, S., L. Qiu, X. Yan, W. Jin, Y. Wang, L. Chen, et al. 2012. Loss of microRNA 122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G(1)-modulated P53 activity. Hepatology 55:730–741.
7. Li, C., Y. Wang, S. Wang, B. Wu, J. Hao, H. Fan, et al. 2013. Hepatitis B virus mRNAmediated miR-122 inhibition upregulates PTTG1-binding protein, which promotes hepatocellular carcinoma tumor growth and cell invasion. J Virol 87:2193–2205.
8. Song, K., C. Han, J. Zhang, D. Lu, S. Dash, M. Feitelson, et al. 2013. Epigenetic regulation of MicroRNA-122 by peroxisome proliferator activated receptor-gamma and hepatitis b virus X protein in hepatocellular carcinoma cells. Hepatology 58:1681–1692.
9. D'Ambrogio, A., W. Gu, T. Udagawa, C. C. Mello, J. D. Richter. 2012. Specific miRNA stabilization by Gld2-catalyzed monoadenylation. Cell Rep 2:1537-45.
10. Peng, F., X. Xiao, Y. Jiang, K. Luo, Y. Tian, M. Peng, et al. 2014. HBx down-regulated Gld2 plays a critical role in HBV-related dysregulation of miR-122. PLoS One 9:e92998.
11. Jopling, C. L, S. Schutz, P. Sarnow. 2008. Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome. Cell Host Microbe 4:77–85.
12. Jangra, R. K., M. Yi, and S. M. Lemon. 2010. Regulation of hepatitis C virus translation and infectious virus production by the microRNA miR-122. J Virol 84: 6615-6625.
13. Machlin, E. S., P. Sarnow, and S. M. Sagan. 2011. Masking the 5' terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex. Proceedings of the National Academy of Sciences of the United States of America 108: 3193-3198.
14. Henke, J. I., D. Goergen, J. Zheng, Y. Song, C. G Schuttler, C. Fehr, et al. 2008. MicroRNA-122 stimulates translation of hepatitis C virus RNA. EMBO J 27:3300–3310.
15. Goergen, D., and M. Niepmann. 2012. Stimulation of Hepatitis C Virus RNA translation by microRNA-122 occurs under different conditions in vivo and in vitro. Virus Res 167:343–352.
16. Wilson, J. A., C. Zhang, A. Huys, C. D. Richardson. 2011. Human Ago2 is required for efficient microRNA 122 regulation of hepatitis C virus RNA accumulation and translation. J Virol 85:2342–2350.
17. Conrad, K.D., F. Giering, C. Erfurth, A. Neumann, C. Fehr, G. Meister, et al. 2013. MicroRNA-122 dependent binding of Ago2 protein to hepatitis C virus RNA is associated with enhanced RNA stability and translation stimulation. PLoS One 8:e56272.
18. Li Y., T. Masaki, D. Yamane, D. R. McGivern, S. M. Lemon. 2013. Competing and noncompeting activities of miR-122 and the 50 exonuclease Xrn1 in regulation of hepatitis C virus replication. Proc Natl Acad Sci U S A
110:1881–1886.
19. Cheung, O., P. Puri, C. Eicken, M. J. Contos, F. Mirshahi, J. W. Maher, et al. 2008. Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression. Hepatology 48:1810–1820.
20. Kutay, H., S. Bai, J. Datta, T. Motiwala, I. Pogribny, W. Frankel, S. T. Jacob, and K. Ghoshal. 2006. Downregulation of miR-122 in the rodent and human hepatocellular carcinomas. Journal of cellular biochemistry 99: 671-678.
21. Tsai, W. C., P. W. Hsu, T. C. Lai, G. Y. Chau, C. W. Lin, C. M. Chen, C. D. Lin, Y. L. Liao, J. L. Wang, Y. P. Chau, M. T. Hsu, M. Hsiao, H. D. Huang and A. P. Tsou. 2009. MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma. Hepatology 49:1571-82.
22. Tsai, W. C., S. D. Hsu, C. S. Hsu, T. C. Lai, S. J. Chen, R. Shen, Y. Huang, H. C. Chen, C. H. Lee, T. F. Tsai, M. T. Hsu, J. C. Wu, H. D. Huang, M. S. Shiao, M. Hsiao, and A. P. Tsou. 2012. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. The Journal of clinical investigation 122: 2884-2897.
23. Hsu, S. H., B. Wang, J. Kota, J. Yu, S. Costinean, H. Kutay, L. Yu, S. Bai, K. La Perle, R. R. Chivukula, H. Mao, M. Wei, K. R. Clark, J. R. Mendell, M. A. Caligiuri, S. T. Jacob, J. T. Mendell, and K. Ghoshal. 2012. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. The Journal of clinical investigation 122: 2871-2883.
24. Bai S, M. W. Nasser, B. Wang, S-H. Hsu, J. Datta, H. Kutay, et al. 2009. MicroRNA-122 inhibits tumorigenic properties of hepatocellular carcinoma cells and sensitizes these cells to sorafenib. J Biol Chem 284:32015–32027.
25. Zeng, C., R. Wang, D. Li, X. J. Lin, Q. K. Wei, Y. Yuan, Q. Wang, W. Chen, and S. M. Zhuang. 2010. A novel GSK-3 beta-C/EBP alpha-miR-122-insulin-like growth factor 1 receptor regulatory circuitry in human hepatocellular carcinoma. Hepatology 52: 1702-1712.
26. Xu, J., X. Zhu, L. Wu, R. Yang, Z. Yang, Q. Wang, and F. Wu. 2012. MicroRNA-122 suppresses cell proliferation and induces cell apoptosis in hepatocellular carcinoma by directly targeting Wnt/β -catenin pathway. Liver Int 32: 752-760.
27. Gramantieri, L., M. Ferracin, F. Fornari, A. Veronese, S. Sabbioni, C. G. Liu, G. A. Calin, C. Giovannini, E. Ferrazzi, G.L. Grazi, C. M. Croce, L. Bolondi, M. Negrini. 2007. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Res 67:6092–9.
28. Fornari, F., L. Gramantieri, C. Giovannini, A. Veronese, M. Ferracin, S. Sabbioni, G. A. Calin, G. L. Grazi, C. M. Croce, S. Tavolari, P. Chieco, M. Negrini, and L. Bolondi. 2009. MiR-122/cyclin G1 interaction modulates p53 activity and affects doxorubicin sensitivity of human hepatocarcinoma cells. Cancer Res 69: 5761-5767.
29. El-Serag, H. B., K. L. Rudolph. 2007. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132:2557–2576.
30. Karin, M., F. R. Greten. 2005. NF-κB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 5:749-59.
31. Maeda, S., H. Kamata, J. L. Luo, H. Leffert, M. Karin. 2005. IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 121:977–990.
32. Levrero, M. 2006. Viral hepatitis and liver cancer: the case of hepatitis C. Oncogene 25: 3834-3847.
33. Maeda, S. 2010. NF-κB, JNK, and TLR Signaling Pathways in Hepatocarcinogenesis. Gastroenterol Res Pract 2010: 367694.
34. He, G., M. Karin. 2011. NF-κB and STAT3 - key players in liver inflammation and cancer. Cell Res 21: 159-168.
35. Li, C., M. Deng, J. Hu, X. Li, L. Chen, Y. Ju, J. Hao, and S. Meng. 2016. Chronic inflammation contributes to the development of hepatocellular carcinoma by decreasing miR-122 levels. Oncotarget 7: 17021-17034.
36. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12: 121-127.
37. Joshi-Barve, S., S. S. Barve, K. Amancherla, L. Gobejishvili, D. Hill, M. Cave, P. Hote, C. J. McClain. 2007. Palmitic acid induces production of proinflammatory cytokine interleukin-8 from hepatocytes. Hepatology 46: 823-830.
38. Harvey, C. E., J. J. Post, P. Palladinetti, A. J. Freeman, R. A. Ffrench, R. K. Kumar, G. Marinos, A. R. Lloyd. 2003. Expression of the chemokine IP-10
(CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation. J Leukoc Biol 74:360-9.
39. Dominguez, M., R. Miquel, J. Colmenero, M. Moreno, J. C. García-Pagán, J. Bosch, V. Arroyo, P. Ginès, J. Caballería, R. Bataller. 2009. Hepatic expression of CXC chemokines predicts portal hypertension and survival in patients with alcoholic hepatitis. Gastroenterology 136:1639-50.
40. Haybaeck, J., N. Zeller, M. J. Wolf, A. Weber, U. Wagner, M. O. Kurrer, J. Bremer, G. Iezzi, R. Graf, P. A. Clavien, R. Thimme, H. Blum, S. A. Nedospasov, K. Zatloukal, M. Ramzan, S. Ciesek, T. Pietschmann, P. N. Marche, M. Karin, M. Kopf, J. L. Browning, A. Aguzzi, and M. Heikenwalder. 2009. A lymphotoxin-driven pathway to hepatocellular carcinoma. Cancer cell 16: 295-308.
41. Dagouassat, M., N. Suffee, H. Hlawaty, O. Haddad, F. Charni, C. Laguillier, R. Vassy, L. Martin, P. O. Schischmanoff, L. Gattegno, O. Oudar, A. Sutton, N. Charnaux. 2010. Monocyte chemoattractant protein-1 (MCP-1)/CCL2 secreted by hepatic myofibroblasts promotes migration and invasion of human hepatoma cells. Int J Cancer 126:1095-108.
42. Chen, T. A., J. L. Wang, S. W. Hung, C. L. Chu, Y. C. Cheng, and S. M. Liang. 2011. Recombinant VP1, an Akt inhibitor, suppresses progression of hepatocellular carcinoma by inducing apoptosis and modulation of CCL2 production. PloS one 6: e23317.
43. Ren, Y, R. T. Poon, H. T. Tsui, W. H. Chen, Z. Li, C. Lau, et al. 2003. Interleukin-8 serum levels in patients with hepatocellular carcinoma: correlations with clinicopathological features and prognosis. Clin Cancer Res 9:5996-6001.
44. Akiba, J, H. Yano, S. Ogasawara, K. Higaki, M. Kojiro. 2001. Expression and function of interleukin-8 in human hepatocellular carcinoma. Int J Oncol 18:257-264.
45. Fujii, H., Y. Itoh, K. Yamaguchi, N. Yamauchi, Y. Harano, T. Nakajima, M. Minami, T. Okanoue. 2004. Chemokine CCL20 enhances the growth of HuH7 cells via phosphorylation of p44/42 MAPK in vitro. Biochem Biophys Res Commun 322:1052-8.
46. Ke-Zhu, H., F. Zhi-Qiang, and G.Hua. 2015. Chemokine ligand 20 enhances progression of hepatocellular carcinoma via epithelial-mesenchymal transition. World J Gastroenterol 21: 475–483.
47. Jiang, R., Z. Tan, L. Deng, Y. Chen, Y. Xia, Y. Gao, X. Wang, B. Sun. 2011. Interleukin-22 promotes human hepatocellular carcinoma by activation of STAT3. Hepatology 54:900-9.
48. Schmidt-Arras, D., S. Rose-John. 2016. IL-6 pathway in the liver: from physiopathology to therapy. J Hepatol 64:1403-15.
49. Reiner, S. L. 2007. Development in motion: helper T cells at work. Cell 129:33-36.
50. Dong, C. 2008. TH17 cells in development: an updated view of their molecular identity and genetic programming. Nat Rev Immunol 8:337-348.
51. Yin, S., Y. Fan, H. Zhang, Z. Zhao, Y. Hao, J. Li, C. Sun, J. Yang, Z. Yang, X. Yang, J. Lu, J. J. Xi. 2016. Differential TGFβ pathway targeting by miR-122 in humans and mice affects liver cancer metastasis. Nat Commun 7:11012.
52. Berasain, C., J. Castillo, M. J. Perugorria, M. U. Latasa, J. Prieto, M. A. Avila. 2009. Inflammation and liver cancer: new molecular links. Annals of the New York Academy of Sciences 1155:206-21.
53. Zeremski, M, L. M. Petrovic, L. Chiriboga, Q. B. Brown, H. T. Yee, M. Kinkhabwala, I. M. Jacobson, R. Dimova, M. Markatou, A. H.Talal. 2008. Intrahepatic levels of CXCR3-associated chemokines correlate with liver
inflammation and fibrosis in chronic hepatitis C. Hepatology 48:1440-50.
54. Richmond, A. Nf-kappa B, chemokine gene transcription and tumour growth. 2002. Nat Rev Immunol 2:664-74.
55. Zhao, L., J. Xia, X. Wang, F. Xu. 2014. Transcriptional regulation of CCL20 expression. Microbes Infect 16:864-70.
56. Yulin, X., J. Z. Chang, Y. Jing, Z. Yan, T. Zhaoxia, K. Xuemei and W. Xiaohong. 2015. Hepatitis C virus represses the cellular antiviral response by upregulating the expression of signal transducer and activator of transcription 3 through sponging microRNA 122. Mol Med Rep 11: 1733–1737.
57. Aomatsu, T., H. Imaeda, K. Takahashi, T. Fujimoto, E. Kasumi, A. Yoden, H. Tamai, Y. Fujiyama, A. Andoh. 2012. Tacrolimus (FK506) suppresses TNF-α-induced CCL2 (MCP-1) and CXCL10 (IP-10) expression via the inhibition of p38 MAP kinase activation in human colonic myofibroblasts. Int J Mol Med 30:1152-8.
58. Wong, C. C., S. L. Au, A. P. Tse, I. M. Xu, R. K. Lai, D. K. Chiu, L. L. Wei, D. N. Fan, F. H. Tsang, R. C. Lo, C. M. Wong, I. O. Ng. 2014. Switching of pyruvate kinase isoform L to M2 promotes metabolic
reprogramming in hepatocarcinogenesis. PLoS One 9:e115036.
59. Chen, T. A., J. L. Wang, S. W. Hung, C. L. Chu, Y. C. Cheng, and S. M. Liang. 2011. Recombinant VP1, an Akt inhibitor, suppresses progression of hepatocellular carcinoma by inducing apoptosis and modulation of CCL2 production. PloS one 6: e23317.
60. Zipin-Roitman, A., T. Meshel, O. Sagi-Assif, B. Shalmon, C.Avivi, R. M. Pfeffer, I. P. Witz, A. Ben-Baruch. 2007. CXCL10 promotes invasion-related properties in human colorectal carcinoma cells. Cancer Res 67:3396-405.
61. Li, X. P., X. Y. Yang, E. Biskup, J. Zhou, H. L. Li, Y. F. Wu, M. L. Chen, F. Xu. 2015. Co-expression of CXCL8 and HIF-1α is associated with metastasis and poor prognosis in hepatocellular carcinoma. Oncotarget 6:22880-9.