臺灣博碩士論文加值系統

肝癌(Hepatocellular carcinoma, HCC) 在癌症死因中位居第二。 HCC的主要病因為HBV、HCV的感染和酒精性肝病， 隨這生活型態的改變在臨床由非酒精性脂肪肝病(non-alcoholic fatty liver disease, NAFLD) 生成的HCC案例逐漸增多。 NAFLD並無明顯病徵，僅有輕微脂肪變性及肝細胞氣球樣變，隨後發展成非酒精性肝炎(non-alcoholic steatohepatitis , NASH)進一步肝硬化肝功能尚失， 最終走向HCC。 MicroRNA-7(miR-7) 為影響腫瘤的發生及發展的關鍵腫瘤抑制者， 先前研究指出ob/ob大鼠肝臟中發現有下調的現象; miR-7會經過標定結合進而調控PI3K/AKT/mTOR pathway已達到抑制腫瘤生成與轉移， Liu et al, (2016) 文獻證實此條訊息途徑和肝臟中的脂肪代謝有密切關聯。 在本篇研究中我們利用L-FABP promoter專一表現miR-7a-spong於斑馬魚肝臟， 偵測miR-7a目標基因CUL5、GSK3β、PIK3CD、mTOR、p70s6k表現量上升。 餵食實驗中高(20%) 及低(<5%)油脂飼養肝臟中表現miR-7a-sp的實驗室組相較於WT對照組肝臟中的油脂累皆積明顯較多，且在成魚切片後觀察到細胞型態的異常及嚴重脂肪肝趨勢，同時也偵測到了肝細胞增生的訊號與PPARα和C/EBPβ mRNA的表現量上升， 透過研究這些現象期望建立一可研究早發性NAFLD之斑馬魚品系，對肝臟疾病的預防及治療有所貢獻。

MicroRNAs (miRNAs) are short non-coding RNAs of 20-24 nucleotides that play key roles in the regulation of gene expression. Recently, several researchers have reported that miRNA we links to the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and the progression to non-alcoholic steatohepatitis (NASH). NASH may progress to advanced-stages of hepatic fibrosis, cirrhosis and liver failure, and then causes of death and hepatocellular carcinoma (HCC). However, there is rarely known about specific therapies for NASH. To development of new target therapy is urgently needed. MicroRNA-7 (miR-7) plays an important role in the occurrence and development of tumors as a key tumor suppressor. Previous research indicated that mir-7a is down-regulated in ob/ob mouse liver and HNF4a-NF-jB feedback circuit including mir-7 is identified in HCC, miR-7 directly inhibits a number of oncogenic targets and impedes cancer progression in vitro and in vivo. Therefore, we designed a miR-7a sponge expression vector drove by liver specific promoter L-FABP combined fluorescent gene to establish transgenic fish. We show that miR-7-silencing activity is specifically knocked down in the transgenic fish liver. As the insights into our research aims for studying NAFLD, feeding these transgenic fish with high fat diet (HFD) and low fat diet (LFD) . We observed the accrual of fat in hepatocytes with Oil Red O staining. Our data shows that miR-7a sponge transgenic fishs have more lipid droplet accumulation in liver than WT with HFD and LFD. In the future, we will plan to analyze miR-7 target genes and of cell proliferation and cell apoptosis in transgenic fish, it can be expected to activities help the study of NAFLD.

謝誌 1
摘要 2
Abstract 3
目錄 4
表目錄 6
圖目錄 7
壹、 序論 1
(一) 動物模式物種─斑馬魚 1
(二) 肝臟的油脂堆積 1
(三) MicroRNAs生源論和作用機制 2
(四) MicroRNAs和代謝 3
(五) MicroRNA-7a and hepatocellular carcinoma 3
(六) Tol2 system 5
(七) MicroRNA sponge 5
(八) 研究動機 6
貳、 材料與方法 7
1. 材料 7
A、生物材料 7
B、反應試劑與製備 7
2. 實驗方法 11
A、 表現載體設計 11
B、 基因轉殖斑馬魚建立 14
C、 高低油脂含量飼料餵食測試 14
D、 Oil-Red O stain 15
E、 基因表現量檢測 15
F、 石蠟切片與H&E染色 16
G、 石蠟切片Proliferating cell nulear antigen (PCNA) 染色 17
參、 實驗結果 19
(一) 建立肝臟特異表現miR-7a-sponge基因轉殖斑馬魚 19
(二) 高低油脂飼料餵食之Oil-Red O染色結果皆顯示miR-7a-sp轉基因魚肝臟油脂累積量高 19
(三) 肝臟中miR-7a功能下降後出現脂肪肝及發炎現象 20
肆、 討論 22
參考文獻 25
伍、表格 30
陸、圖 33
柒、附圖 46

Alves RC, Alves D, Guz B, Matos C, Viana M, Harriz M, Terrabuio D, Kondo M, Gampel O, Polletti P. (2011) Advanced hepatocellular carcinoma. Review of targeted molecular drugs. Ann Hepatol. 2011;10:21-27.
Bravo-Egana V, Rosero S, Molano RD, Pileggi A, Ricordi C, Domínguez-Bendala J, Pastori RL. (2008)Quantitative differential expression analysis reveals miR-7 as major islet microRNA. Biochem Biophys Res Commun. 2008;366:922-926.
Chou, Y.T.; Lin, H.H.; Lien, Y.C.; Wang, Y.H.; Hong, C.F.; Kao, Y.R.; Lin, S.C.; Chang, Y.C.; Lin, S.Y.; Chen, S.J.; et al. (2010) Egfr promotes lung tumorigenesis by activating mir-7 through a ras/erk/myc pathway that targets the ets2 transcriptional repressor erf. Cancer Res. 2010, 70, 8822–8831.
Correa-Medina M, Bravo-Egana V, Rosero S, Ricordi C, Edlund H, Diez J, Pastori RL. MicroRNA miR-7 is preferentially expressed in endocrine cells of the developing and adult human pancreas. Gene Expr Patterns. 2009;9:193-199.
Csaki LS, Reue K. (2010) Lipins: multifunctional lipid metabolism proteins. Annu Rev Nutr. 2010;30:257–272. doi: 10.1146/annurev.nutr.012809.104729.
Chattopadhyay M, Selinger ES, Ballou LM, Lin RZ. 2011 Ablation of PI3K p110-α prevents high-fat diet-induced liver steatosis. Diabetes. 2011 May;60(5):1483-92. doi: 10.2337/db10-0869.
Calvisi DF1, Wang C, Ho C, Ladu S, Lee SA, Mattu S, Destefanis G, Delogu S, Zimmermann A, Ericsson J, Brozzetti S, Staniscia T, Chen X, Dombrowski F, Evert M. 2011. Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma. Gastroenterology. 2011 Mar;140(3):1071-83. doi: 10.1053/j.gastro.
D. D. Liu, C. C. Han, H. F. Wan, F. He, H. Y. Xu, S. H. Wei, X. H. Du and F. Xu (2016) Effects of inhibiting PI3K-Akt-mTOR pathway on lipid metabolism
Davis S, Lollo B, Freier S, Esau C (2006). Improved targeting of miRNA with antisense oligonucleotides. Nucleic Acids Res 34: 2294–2304
Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ. (2005) Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest. 2005;115:1343–1351.
Dooley, K., and Zon, L.I. (2000). Zebrafish: a model system for the study of human disease. Current opinion in genetics & development 10, 252-256
D. D. Liu, C. C. Han†, H. F. Wan, F. He, H. Y. Xu, S. H. Wei, X. H. Du and F. Xu (2016) Effects of inhibiting PI3K-Akt-mTOR pathway on lipid metabolism. The Animal Consortium 2016doi:10.1017/S1751731116000380.
Evans SL, Schön A, Gao Q, Han X, Zhou X, Freire E, Yu XF (2014). HIV-1 Vif N-terminal motif is required for recruitment of Cul5 to suppress APOBEC3. Retrovirology. 2014 Jan 14;11:4. doi: 10.1186/1742-4690-11-4.
Fan CY1, Pan J, Usuda N, Yeldandi AV, Rao MS, Reddy JK.(1998) Steatohepatitis, spontaneous peroxisome proliferation and liver tumors in mice lacking peroxisomal fatty acyl-CoA oxidase. Implications for peroxisome proliferator-activated receptor alpha natural ligand metabolism. J Biol Chem. 1998 Jun 19;273(25):15639-45.
Fang Y, Xue JL, Shen Q, Chen J, Tian L. (2012) MicroRNA-7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3-kinase/Akt pathway in hepatocellular carcinoma. Hepatology. 2012;55:1852-1862.
Fang Y, Xue JL, Shen Q, Chen J, Tian L. (2012)MicroRNA-7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3-kinase/Akt pathway in hepatocellular carcinoma. Hepatology. 2012;55:1852-1862.
Frank J. Gonzalez, Jeffrey M. Peters, Russell C. Cattley (1998) Mechanism of Action of the Nongenotoxic PeroxisomeProliferators: Role of the Peroxisome Proliferator-Activated Receptora. Journal of the National Cancer Institute, Vol. 90, No. 22, November 18
Giles, K.M.; Brown, R.A.; Epis, M.R.; Kalinowski, F.C.; Leedman, P.J. (2013)Mirna-7-5p inhibits melanoma cell migration and invasion. Biochem. Biophys. Res. Commun. 2013, 430, 706–710.
HARALD SCHREM, JÜRGEN KLEMPNAUER, AND JÜRGEN BORLAK (2004) Liver-Enriched Transcription Factors in LiverFunction and Development. Part II: the C/EBPs and DSite-Binding Protein in Cell Cycle Control,Carcinogenesis, Circadian Gene Regulation, LiverRegeneration, Apoptosis, and Liver-Specific GeneRegulation doi: 10.1124/pr.56.2.5
homeostasis in goose primary hepatocytes. Animal, page 1 of 9doi:10.1017/S1751731116000380
Janardan K. Reddy and M. Sambasiva Rao (2006) Lipid Metabolism and Liver Inflammation. II. Fatty liver disease and fatty acid oxidation. Am J Physiol Gastrointest Liver Physiol 290: G852–G858, 2006
Kalinowski, F.C.; Giles, K.M.; Candy, P.A.; Ali, A.; Ganda, C.; Epis, M.R.; Webster, R.J.; Leedman, P.J. (2012)Regulation of epidermal growth factor receptor signaling and erlotinib sensitivity in head and neck cancer cells by mir-7. PLoS One 2012, 7, e47067.
Kari, G., Rodeck, U., and Dicker, A.P. (2007). Zebrafish: an emerging model system for human disease and drug discovery. Clinical pharmacology and therapeutics 82, 70-80.
Kefas, B.; Godlewski, J.; Comeau, L.; Li, Y.; Abounader, R.; Hawkinson, M.; Lee, J.; Fine, H.; Chiocca, E.A.; Lawler, S.; et al. (2008)Microrna-7 inhibits the epidermal growth factor receptor and the akt pathway and is down-regulated in glioblastoma. Cancer Res. 2008, 68, 3566–3572
Kei Miyakawa, Satoko Matsunaga, Kazuhiko Kanou, Atsushi Matsuzawa, Ryo Morishita, Ayumi Kudoh, Keisuke Shindo, Masaru Yokoyama, Hironori Sato, Hirokazu Kimura, Tomohiko Tamura, Naoki Yamamoto, Hidenori Ichijo, Akifumi Takaori-Kondo & Akihide Ryo (2015) ASK1 restores the antiviral activity of APOBEC3G by disrupting HIV-1 Vif-mediated counteraction. Nature Communications 6, Article number: 6945 doi:10.1038/ncomms7945
Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M (2005). Silencing of microRNAs in vivo with ‘antagomirs’. Nature 438: 685–689
Kudo M.(2012) Signaling pathway/molecular targets and new targeted agents under development in hepatocellular carcinoma. World J Gastroenterol. 2012;18:6005-6017.
Kudo Y, Tanaka Y, Tateishi K, Yamamoto K, Yamamoto S, Mohri D, Isomura Y, Seto M, Nakagawa H, Asaoka Y, Tada M, Ohta M, Ijichi H, Hirata Y, Otsuka M, Ikenoue T, Maeda S, Shiina S, Yoshida H, Nakajima O, Kanai F, Omata M, Koike K (2011). Altered composition of fatty acids exacerbates hepatotumorigenesis during activation of the phosphatidylinositol 3-kinase pathway. J Hepatol. 2011 Dec;55(6):1400-8. doi: 10.1016/j.jhep
LU Wan, YANG Renqiang, WANG Ling (2013) Research progress of exosomes. 《生命的化学》2013年33卷4期
Ma C, Qi Y, Shao L, Liu M, Li X, Tang H.(2013) Downregulation of miR-7 upregulates Cullin 5 (CUL5) to facilitate G1/S transition in human hepatocellular carcinoma cells. IUBMB Life. 2013;65:1026-1034.
Margaret S. Ebert and Phillip A. Sharp (2010) MicroRNA sponges: Progress and possibilities. RNA. 2010 Nov; 16(11): 2043–2050.
Martin D. Jansson, Anders H. Lund (2012) MicroRNA and cancer. MOLECULARON COLOGY6 (2012) 590-610
Meister G, Landthaler M, Dorsett Y, Tuschl T (2004). Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10: 544–550
Meza-Sosa, K.F.; Perez-Garcia, E.I.; Camacho-Concha, N.; Lopez-Gutierrez, O.; Pedraza-Alva, G.; Perez-Martinez, L.(2014) Mir-7 promotes epithelial cell transformation by targeting the tumor suppressor klf4. PLoS One 2014, 9, e103987.
Moore KJ1, Rayner KJ, Suárez Y, Fernández-Hernando C (2010) microRNAs and cholesterol metabolism. Trends Endocrinol Metab. 2010 Dec;21(12):699-706.
Naomoto Harada, Zenjun Oda, Yoshikazu Hara, Koji Fujinami, Mayumi Okawa, Katsuya Ohbuchi, Mari Yonemoto, Yuika Ikeda, Kenji Ohwaki, Katsumi Aragane, Yoshitaka Tamai, and Jun Kusunoki (2007). Hepatic De Novo Lipogenesis Is Present in Liver-Specific ACC1-Deficient Mice. Mol Cell Biol. 2007 Mar; 27(5): 1881–1888.
Nguyen HT, Dalmasso G, Yan Y, Laroui H, Dahan S, Mayer L, Sitaraman SV, Merlin D. (2010)MicroRNA-7 modulates CD98 expression during intestinal epithelial cell differentiation. J Biol Chem. 2010;285:1479-1489.
Ning, B.F.; Ding, J.; Liu, J.; Yin, C.; Xu, W.P.; Cong, W.M.; Zhang, Q.; Chen, F.; Han, T.; Deng, X.; et al. Hepatocyte nuclear factor 4alpha-nuclear factor-kappab feedback circuit modulates liver cancer progression. Hepatology 2014, 60, 1607–1619.
Ørom UA, Kauppinen S, Lund AH (2006). LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene 10: 137–141
Pharmacological Reviews June 2004 vol. 56 no. 2 291-330.
Rai, K.; Takigawa, N.; Ito, S.; Kashihara, H.; Ichihara, E.; Yasuda, T.; Shimizu, K.; Tanimoto, M.; Kiura, K.(2011)Liposomal delivery of microrna-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells. Mol. Cancer Ther. 2011, 10, 1720–1727.
Reddy, S.D.; Ohshiro, K.; Rayala, S.K.; Kumar, R.(2008) Microrna-7, a homeobox d10 target, inhibits p21-activated kinase 1 and regulates its functions. Cancer Res. 2008, 68, 8195–8200.
Tilg and Moschen, (2008) Inflammatory mechanisms in the regulation of insulin resistance. Mol Med. 2008 Mar-Apr; 14(3-4): 222–231.
Wang, Y., Goulart, R.A., and Pantanowitz, L. (2011). Oil red O staining in cytopathology. Diagnostic cytopathology 39, 272-273
Wan-Qun Chen, Ling Hu, Geng-Xin Chen, Hai-Xia Deng(2015) Role of microRNA-7 in digestive system malignancy. Published online Jan 15, 2016. doi: 10.4251/wjgo.v8.i1.121
Yoshio Sumida, Atsushi Nakajima, Hideyuki Hyogo, Saiyu Tanaka, Masafumi Ono, Hideki Fujii, Yuichiro Eguchi, Masato Yoneda, (2015) Non-invasive scoring systems for predicting NASH in Japan: evidences from Japan Study Group of NAFLD. Integr Mol Med Volume 2(2): 145-149.
Zhang X, Hu S, Zhang X, Wang L, Zhang X, Yan B, Zhao J, Yang A, Zhang R. (2014)MicroRNA-7 arrests cell cycle in G1 phase by directly targeting CCNE1 in human hepatocellular carcinoma cells. Biochem Biophys Res Commun. 2014;443:1078-1084.
Zhang Y, Liu D, Chen X, et al. Secreted monocytic miR-150 enhances targeted endothelial cell migration. Mol Cell, 2010, 39: 133-144
Zhang, N.; Li, X.; Wu, C.W.; Dong, Y.; Cai, M.; Mok, M.T.; Wang, H.; Chen, J.; Ng, S.S.; Chen, M.; et al. (2013)Microrna-7 is a novel inhibitor of yy1 contributing to colorectal tumorigenesis. Oncogene 2013, 32, 5078–5088.