Action mechanisms of IGF2b and miR-122 in zebrafish liver and human hepatocellular carcinoma cells

• Main Authors: Ji-Fan Lin, 林致凡
• Other Authors: Jen-Leih Wu
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/47801093546600277901

博士 === 國防醫學院 === 生命科學研究所 === 96 === Increased expression of insulin-like growth factor II (IGF2) has been well documented in human hepatitis, hepatoma and hepatocellular carcinoma (HCC). Recent microarray study demonstrated elevated IGF2 level in human steatosis. However, IGF2 is the causative agent or just ensures maintenance of proliferation of neoplastic cells as a secondary signal remains unclear. Utilizing the previously established GFP-liver transgenic zebrafish, in which changes in liver morphology are easily observable, the zebrafish overexpressing IGF2b, functional homologue of human IGF2, in hepatocytes was generated to analyze the effect of deregulated IGF2 signaling on liver homeostasis. After hatching, these zebrafish larvae rapidly developed hepatomegaly, with a liver volume 1.5 times larger than that of control larvae. The hepatomegaly resulted from imbalanced homeostasis through up-regulation of cell-cycle (Cyclin D1), anti-apoptosis (Bcl-xL), and liver-enriched transcription factors (HNF1, HNF4 and C/EBP) and down-regulation of IGF binding proteins (IGFBP1 and IGFBP3) and apoptosis markers (bax). Furthermore, Akt activation and up-regulation of lipogenesis-related genes such as C/EBP, PPAR, SREBP1, ACC, SCD-1, and FASN were revealed in the enlarged livers, which led to steatosis. The same genes profile was also observed in transgenic zebrafish with liver-specific expression of IGF2b. The mRNA levels of -catenin down stream signals, c-myc and cyclin D1, were also increased in IGF2b-overexpressing livers, which indicates a high incidence of tumor. These data demonstrate the importance and initiation role of elevated IGF2 level in the progression of liver disease. The transient transgenic zebrafish larvae may serve as a potential model to facilitate the screening of inhibitory compounds in the IGF2 signaling pathway. Additionally, liver-specific microRNA (miRNA), miR-122, was found to regulate lipid and cholesterol metabolism. Inhibition of miR-122 levels in vivo by administration of a modified-oligonucleotides results in reduced serum lipid and cholesterol. miR-122 is frequently down-regulated in human hepatocellular carcinoma (HCC). In an effort to identify novel miR-122 targets that related to the formation of steatosis or HCC, in silico analysis detected that it has a putative binding site in the Bcl-w 3’-untranslated region (UTR). In the HCC-derived cell lines Hep3B and HepG2, miR-122 modulates Bcl-w expression by directly targeting binding site within the 3’-UTR was then demonstrated. The cellular mRNA and protein levels of Bcl-w were repressed by elevated levels of miR-122, which subsequently led to reduction of cell viability and activation of caspase-3. Thus, Bcl-w is a direct target of miR-122 that functions as an endogenous apoptosis regulator in an HCC-derived cell lines. Taken together, these studies revealed that IGF2b plays an important role in the formation of steatosis by activating lipid biogenesis enzymes and constitutive stimulation of IGF signaling pathway via AKT/PI3 pathway. The post-transcriptional inhibition of Bcl-w by miR-122 demonstrated the decreased levels of miR-122 in HCC may contribute to anti-apoptotic and dysregulated growth control of cancerous cells.