Objective:Intratumoral administration of adenoviral vector encoding herpes simplex virus(HSV)thymidine kinase(TK)gene(Ad-TK)followed by systemic ganciclovir(GCV)is an effective approach in treating experimental hepatocellular carcinoma(HCC).However,hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy.miR-122 is an abundant,liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines.These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation.Methods:By inserting miR-122 target sequences(miR-122T)in the 3'untranslated region(UTR)of TK gene,we constructed adenovirus(Ad)vectors expressing miR-122-regulated TK(Ad-TK-122T)and report genes.After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model,we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T.Results:Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment.Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo,resulting in an 11-fold improvement of tumor-specific transgene expression.Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor.The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy.Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose,no liver damage was found in Ad-TK-122T group.Conclusions:miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.
Gang WangXiaoyan DongWenhong TianYue LuJianyan HuYunfan LiuJie YuchiXiaobing Wu
用我们建立的活细胞miRNA活性谱检测技术测定BHK21、HEK293和Vero三种肾来源细胞系中58种miRNA活性谱,发现miR-206在BHK21细胞中具有特征性高活性。鉴于miR-206是典型的横纹肌特征性miRNA,进一步以小鼠成肌细胞C2C12及人胚肾细胞HEK293为对照,检测了BHK21细胞中miR-206的活性及表达水平。之后,用马血清诱导培养BHK21细胞,检测诱导前后BHK21细胞中骨骼肌肌球蛋白重链(Slowskeletal myosin heavy chain,MHC)表达情况,miR-206活性和表达水平以及受miR-206负调控的Connexin43(Cx43)的表达水平。结果发现,miR-206在BHK21细胞中活性和表达水平都明显高于C2C12细胞;马血清诱导后,BHK21细胞中MHC表达水平升高,miR-206活性和表达水平都升高,而Cx43表达水平下降。结果提示BHK21细胞具有成肌细胞特性。本研究首次发现BHK21细胞中miR-206的高活性,从miRNA角度证实了BHK21细胞来源于肾间质细胞,而不是肾实质细胞。研究结果还提示BHK21细胞有可能作为一种体外模型用于miR-206的功能研究。
