| Summary: | 碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 99 === IGF2 mRNA-binding protein 2 (IGF2BP2/IMP2) was expressed in fetal liver but little in adult liver while overexpressed in the human liver tumors including hepatocellular carcinoma and cholangiocarcinoma of human. To study the molecular mechanism of IMP2 during the progression of liver tumors, transgenic zebrafish lines Tg(fabp10:tetoff; tre:imp2/gfp) with constitutively zebrafish IMP2 and GFP-overexpressed in the liver had been established in our laboratory by using liver-specific LFABP promoter combined with TetOff bi-directional expression system via Tol2 transposon-mediated transgenesis. The liver-specific IMP2 transgenic zebrafish stably inherited to F5 generation will develop severe intrahepatic cholangiocarcinoma (ICC) at around 10-week old. Pathological progression of ICC in IMP2 transgenic zebrafish was revealed from liver injury without cholangiocyte proliferation at 4-week old to cholangiocyte proliferation at 6-week old then progressing to ICC formation at around 10-week old zebrafish. Transgenic IMP2 expression was turned off by tetracycline treatment (1μg/ml) from 1-week to 6-week old. There was no intrahepatic cholangiocyte proliferation observed in IMP2 transgenic zebrafish treated with tetracycline at 6-week old. The result confirmed that intrahepatic cholangiocyte proliferation in IMP2 transgenic zebrafish is an IMP2-dependent phenotype. To study the early stage mechanism of IMP2-induced ICC, the liver with abnormal hepatocytes of 4-week IMP2 transgenic fish was compared with normal liver of control transgenic fish Tg(fabp10:tetoff; tre:gfp) by microarray analysis and evaluated by Q-PCR.
We found the critical antocrine/paracrine signaling genes including IGF-IIa, IGF-2b, IGF-IRa, IGF-IRb and Progranulin A (PGRN-A) were all up-regulated in the liver of 4-week IMP2 transgenic zebrafish. The results indicate liver-specific IMP2 overexpression may activate IGF-II and progranulin signalling to stimulate cholangiocyte proliferation and increase PTEN and AKT phosphorylation to attenuate PTEN and activate AKT-mediated anti-apoptosis signal in the early stage of ICC progression. Furthermore, the antisense vivo-Morpholinos of IGF-2b, IGF-IRa, IGF-IRb and PGRN-A were used to treat IMP2 transgenic zebrafish from 3-week to 6-week old by intraperitoneal (I.P.) injection, respectively. Inhibition of IGF-IRa and IGF-IRb by vivo-Morpholino (Mo) can significantly reduce intrahepatic cholangiocyte proliferation from 78% (control: PBS injection) to 50% and 44%, respectively. Inhibition of IGF-II and PGRN-A by vivo-Mophonino can also reduce intrahepatic cholangiocyte proliferation from 78% to 64% and 75%, respectively. The data indicates that IGF-IRs play a critical role in cholangiocyte proliferation during the early stage of ICC progression induced by IMP2 overexpression. Effective blockage of IGF-IR signaling with vivo-Mophonino may provide the evidence of using IGF-IR signaling as therapeutic target for early diagnosis and/or treatment of ICC. This liver-specific IMP2-induced ICC transgenic zebrafish provides a novel animal model to study the interaction of hepatocytes and cholangiocytes in ICC progression.
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