title: α-Fetoprotein promoter-driven Cre/LoxP-switched RNA interference for hepatocellular carcinoma tissue-specific target therapy.
abstract: BACKGROUND
  RNA 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.
  
  
  METHODS
  Two 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  An 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.
authors.affiliation: Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, PR China.
authors.forename: Yuan-Fei
authors.initials: YF
authors.lastname: Peng
authors.forename: Ying-Hong
authors.initials: YH
authors.lastname: Shi
authors.forename: Zhen-Bin
authors.initials: ZB
authors.lastname: Ding
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.forename: Shuang-Jian
authors.initials: SJ
authors.lastname: Qiu
authors.forename: Bo
authors.initials: B
authors.lastname: Hui
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Gu
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Wei-Ren
authors.initials: WR
authors.lastname: Liu
authors.forename: Jia
authors.initials: J
authors.lastname: Fan
mesh_terms: D000818:Animals; D000071187:Autophagy-Related Protein 5; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D015972:Gene Expression Regulation, Neoplastic; D023061:Gene Order; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009536:Niacinamide / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology; D009928:Organ Specificity / Q000235:genetics; D010671:Phenylurea Compounds / Q000008:administration & dosage / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D000077157:Sorafenib; D047368:Tumor Burden / Q000187:drug effects / Q000235:genetics; D000509:alpha-Fetoproteins / Q000235:genetics*
chemical_list: C495773:Atg5 protein, mouse; D000071187:Autophagy-Related Protein 5; D008869:Microtubule-Associated Proteins; D010671:Phenylurea Compounds; D000509:alpha-Fetoproteins; D009536:Niacinamide; D000077157:Sorafenib