Abstract
BackgroudRNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.MethodsTwo different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.ResultsThe AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.ConclusionsAn efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.
Highlights
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related death [1]
GFP reporter assay showed that the AFP-promoter could efficiently drive GFP expression in AFPproducing HCC cells (HepG2, Hep3B, and HCCLM3) but not in non-HCC cells (L-02, SW1116, and Hela) (Fig. 1B,D)
We examined whether the AFP-Cre/LoxP-short hairpin RNA (shRNA)-Atg5 could sensitize HCC cells to sorafenib treatment
Summary
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related death [1]. The prognosis of HCC is poor for most of patients because HCC is often diagnosed at a late stage and current treatment options are rather limited. The inherent difficulty of treating this malignancy has prompted many to consider new therapeutic approach. In recent years RNA interference (RNAi) has emerged as a potential treatment modality for cancer therapy. RNAi is a sequence-specific posttranscriptional gene silencing process. RNAi has been rapidly developed into a powerful technique for both therapeutic and gene functional research
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