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MicroRNA-122-regulated TK gene effectively inhibits liver toxicity of TK/GCV therapy system
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Abstract:
Objective:o investigate the regulatory effect of liver-specific microRNA-122(miR-122) on the expression of exogenous genes and its role in decreasing the liver toxicity of herpes simplex virus type 1-thymidine kinase/gancyclovir (TK/GCV) therapy system. Methods: MiR-122-regulated pEGFP-122T, pLuc-122T or pTK-122T plasmids were constructed by inserting miR-122 targeting sequence into the 3′-untranslated region (3′-UTR) of the corresponding genes, and the plasmids were then transfected into miR-122-positive Huh7 and miR-122-negative HeLa cells, then the expression of reporter genes and the hepatotoxicity of TK/GCV system were observed. After hydrodynamic delivery of relevant plasmids into mouse liver, the expressions of EGFP and Luc were analyzed by fluorescence microscopy and in vivo bioluminescent imaging; and the hepatotoxicity of TK/GCV system on mice was evaluated by serum ALT, weight, survival and hepatic histology. Results: In Huh7 cells, the EGFP expression and Luc activity were inhibited by miR-122 targeting sequence insertion, and pTK-122T-transfected Huh7 cells showed resistance to cytotoxicity of TK/GCV system; whereas in HeLa cells, the expression of reporter genes and the cytotoxicity of TK/GCV system were not influenced. In vivo results showed that hepatocytes in pEGFP-122T-treated mice did not express EGFP, but 30% hepatocytes in pEGFP-treated mice expressed high level of EGFP; the Luc activity was significantly down-regulated in pLuc-122T-treated mice compared to pLuc-treated mice. Hydrodynamic injection of pTK-122T caused no increase of serum ALT level or decrease of body weight, liver toxicity of GCV treatment, while significant increase of serum ALT level, decrease of body weight and severe liver damages were found in the pTK-treated mice. Conclusion: MiR-122 targeting sequence insertion can inhibit the expression of exogenous genes in the liver, and can effectively decrease the hepatotoxicity of TK/GCV system.
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Project Supported:
Project supported by the Special Key Program of Sceience and Technology of China (No.2008ZX10002-023, No.2008ZX10001-012), and the Self-selected Research Program from National Key Laboratory of Molecular Virology and Genetic Engineering (No.2008-S-0003)
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