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α-Fetoprotein promoter-driven Cre/LoxP-switched RNA interference for hepatocellular carcinoma tissue-specific target therapy.

Peng YF, Shi YH, Ding ZB, Zhou J, Qiu SJ, Hui B, Gu CY, Yang H, Liu WR, Fan J - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, PR China.

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.

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Related in: MedlinePlus

Effectiveness and tissue-specificity of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi.(A) Construction of the AFP-Cre/LoxP-shRNA system which consists of AFP-Cre and LoxP-shRNA vectors. (B) Schematic illustration of the AFP-Cre/LoxP-shRNA system. When the AFP-Cre and LoxP-shRNA vectors co-infect target cells, the AFP promoter drives the downstream Cre recombinase gene expression which subsequently cuts the LoxP-CMV-eGFP-LoxP inside the U6 promoter and activates it. The activated U6 promoter then drives the downstream shRNA expression. The LoxP-shRNA harbors a CMV-eGFP indicator which can indicate the RNAi expression (GFP fluorescence will diminish if the AFP promter initiates the RNAi expression). (C, D) CMV-eGFP indicator and gene silencing analysis by Q-PCR and western blot showed that the AFP-Cre/LoxP-shRNA system targeting Beclin 1 (AFP-Cre/LoxP-shRNA-Beclin1) could efficiently silence target gene (Beclin 1) in a HCC-specific manner. The GFP fluorescence diminished in HCC cells (HCCLM3, HepG2, and Hep3B) but not in non-HCC cells (L-02, Hela, and SW1116) after infection with the AFP-Cre/LoxP-shRNA-Beclin1. Q-PCR and western blot analysis were exemplified by HCC cell line HCCLM3 and non-HCC cell line L-02.
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pone-0053072-g003: Effectiveness and tissue-specificity of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi.(A) Construction of the AFP-Cre/LoxP-shRNA system which consists of AFP-Cre and LoxP-shRNA vectors. (B) Schematic illustration of the AFP-Cre/LoxP-shRNA system. When the AFP-Cre and LoxP-shRNA vectors co-infect target cells, the AFP promoter drives the downstream Cre recombinase gene expression which subsequently cuts the LoxP-CMV-eGFP-LoxP inside the U6 promoter and activates it. The activated U6 promoter then drives the downstream shRNA expression. The LoxP-shRNA harbors a CMV-eGFP indicator which can indicate the RNAi expression (GFP fluorescence will diminish if the AFP promter initiates the RNAi expression). (C, D) CMV-eGFP indicator and gene silencing analysis by Q-PCR and western blot showed that the AFP-Cre/LoxP-shRNA system targeting Beclin 1 (AFP-Cre/LoxP-shRNA-Beclin1) could efficiently silence target gene (Beclin 1) in a HCC-specific manner. The GFP fluorescence diminished in HCC cells (HCCLM3, HepG2, and Hep3B) but not in non-HCC cells (L-02, Hela, and SW1116) after infection with the AFP-Cre/LoxP-shRNA-Beclin1. Q-PCR and western blot analysis were exemplified by HCC cell line HCCLM3 and non-HCC cell line L-02.

Mentions: As the AFP-miRNA required a high titer which was cytotoxic to target cells, it was not desirable for target therapy because it might cause damage to the adjacent normal cells and tissues. Therefore, another system (AFP-Cre/LoxP-shRNA) was constructed (Fig. 3A).


α-Fetoprotein promoter-driven Cre/LoxP-switched RNA interference for hepatocellular carcinoma tissue-specific target therapy.

Peng YF, Shi YH, Ding ZB, Zhou J, Qiu SJ, Hui B, Gu CY, Yang H, Liu WR, Fan J - PLoS ONE (2013)

Effectiveness and tissue-specificity of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi.(A) Construction of the AFP-Cre/LoxP-shRNA system which consists of AFP-Cre and LoxP-shRNA vectors. (B) Schematic illustration of the AFP-Cre/LoxP-shRNA system. When the AFP-Cre and LoxP-shRNA vectors co-infect target cells, the AFP promoter drives the downstream Cre recombinase gene expression which subsequently cuts the LoxP-CMV-eGFP-LoxP inside the U6 promoter and activates it. The activated U6 promoter then drives the downstream shRNA expression. The LoxP-shRNA harbors a CMV-eGFP indicator which can indicate the RNAi expression (GFP fluorescence will diminish if the AFP promter initiates the RNAi expression). (C, D) CMV-eGFP indicator and gene silencing analysis by Q-PCR and western blot showed that the AFP-Cre/LoxP-shRNA system targeting Beclin 1 (AFP-Cre/LoxP-shRNA-Beclin1) could efficiently silence target gene (Beclin 1) in a HCC-specific manner. The GFP fluorescence diminished in HCC cells (HCCLM3, HepG2, and Hep3B) but not in non-HCC cells (L-02, Hela, and SW1116) after infection with the AFP-Cre/LoxP-shRNA-Beclin1. Q-PCR and western blot analysis were exemplified by HCC cell line HCCLM3 and non-HCC cell line L-02.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585287&req=5

pone-0053072-g003: Effectiveness and tissue-specificity of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi.(A) Construction of the AFP-Cre/LoxP-shRNA system which consists of AFP-Cre and LoxP-shRNA vectors. (B) Schematic illustration of the AFP-Cre/LoxP-shRNA system. When the AFP-Cre and LoxP-shRNA vectors co-infect target cells, the AFP promoter drives the downstream Cre recombinase gene expression which subsequently cuts the LoxP-CMV-eGFP-LoxP inside the U6 promoter and activates it. The activated U6 promoter then drives the downstream shRNA expression. The LoxP-shRNA harbors a CMV-eGFP indicator which can indicate the RNAi expression (GFP fluorescence will diminish if the AFP promter initiates the RNAi expression). (C, D) CMV-eGFP indicator and gene silencing analysis by Q-PCR and western blot showed that the AFP-Cre/LoxP-shRNA system targeting Beclin 1 (AFP-Cre/LoxP-shRNA-Beclin1) could efficiently silence target gene (Beclin 1) in a HCC-specific manner. The GFP fluorescence diminished in HCC cells (HCCLM3, HepG2, and Hep3B) but not in non-HCC cells (L-02, Hela, and SW1116) after infection with the AFP-Cre/LoxP-shRNA-Beclin1. Q-PCR and western blot analysis were exemplified by HCC cell line HCCLM3 and non-HCC cell line L-02.
Mentions: As the AFP-miRNA required a high titer which was cytotoxic to target cells, it was not desirable for target therapy because it might cause damage to the adjacent normal cells and tissues. Therefore, another system (AFP-Cre/LoxP-shRNA) was constructed (Fig. 3A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Liver Surgery, Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, PR China.

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.

Show MeSH
Related in: MedlinePlus