摘要
目的:探讨利用肝脏特异性microRNA-122(miR-122)调控外源基因在肝细胞的表达,减轻单纯疱疹病毒1型胸苷激酶和更昔洛韦(herpes simplex virus type 1-thymidine kinase/gancyclovir,TK/GCV)治疗系统的肝毒性。方法:通过在相应基因的3′非翻译区(3′UTR)插入miR-122靶序列,构建miR-122调控的pEGFP-122T、pLuc-122T和pTK-122T表达载体,分别转染miR-122阳性的Huh7肝癌细胞和miR-122阴性的HeLa宫颈癌细胞,观察细胞中报告基因的表达及TK/GCV的细胞毒杀伤作用。水动力法分别注射上述质粒至小鼠体内,冰冻切片和活体成像观察肝脏EGFP和Luc的表达,通过小鼠血清ALT、体质量变化、存活率和肝脏病理变化评价TK/GCV系统的肝毒性。结果:在Huh7细胞,miR-122靶序列的插入抑制了EGFP的表达和Luc的活性,减轻TK/GCV的毒性杀伤;而在HeLa细胞,miR-122靶序列的插入对报告基因表达和TK/GCV的杀伤无影响。体内实验结果显示,pEGFP-122T处理组的肝细胞不表达EGFP,而pEGFP处理组的肝细胞有30%高表达EGFP;与pLuc处理组相比,pLuc-122T处理组的Luc活性下调了33.70%。pTK处理组小鼠血清ALT显著升高、体质量进行性下降、肝脏出现明显病理损伤,进而引起小鼠死亡;而pTK-122T处理组小鼠血清ALT正常、无体质量下降、肝脏切片未见病理改变。结论:miR-122靶序列的插入可抑制外源基因在肝脏的表达,并可有效地降低TK/GCV系统的肝毒性。
Objective:To 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.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2010年第5期492-498,共7页
Chinese Journal of Cancer Biotherapy
基金
国家科技重大专项基金资助项目(No.2008ZX10002-023
No.2008ZX10001-012)
病毒基因工程国家重点实验室自主课题项目(No.2008-S-0003)~~