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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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Activity and tissue-specificity of the AFP promoter.(A) Construction of recombinant lentiviral vectors containing GFP reporter gene driven by AFP promoter or CMV promoter (AFP-GFP and CMV-GFP) for AFP promoter assay. (B,D) The AFP promoter is active and HCC tissue-specific. GFP was highly expressed in AFP-producing HCC cells (HepG2, Hep3B, and HCCLM3) but not in non-HCC cells (normal hepacyte L-02, cervical cancer cell Hela, and colon cancer cell SW1116). (C,E) The AFP promoter was efficient for transgenic expression but its activity was weaker than CMV-promoter, as exemplified by HCCLM3 cells infected with AFP-GFP (MOI = 5–80) and CMV-GFP (MOI = 5).
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pone-0053072-g001: Activity and tissue-specificity of the AFP promoter.(A) Construction of recombinant lentiviral vectors containing GFP reporter gene driven by AFP promoter or CMV promoter (AFP-GFP and CMV-GFP) for AFP promoter assay. (B,D) The AFP promoter is active and HCC tissue-specific. GFP was highly expressed in AFP-producing HCC cells (HepG2, Hep3B, and HCCLM3) but not in non-HCC cells (normal hepacyte L-02, cervical cancer cell Hela, and colon cancer cell SW1116). (C,E) The AFP promoter was efficient for transgenic expression but its activity was weaker than CMV-promoter, as exemplified by HCCLM3 cells infected with AFP-GFP (MOI = 5–80) and CMV-GFP (MOI = 5).

Mentions: A recombinant lentiviral vector containing GFP reporter gene driven by AFP promoter was constructed (AFP-GFP) (Fig. 1A). The activity and tissue-specificity of the AFP promoter were assayed by infecting the AFP-producing and non-AFP-producing cells with the AFP-GFP vector. GFP reporter assay showed that the AFP-promoter could efficiently drive GFP expression in AFP-producing HCC cells (HepG2, Hep3B, and HCCLM3) but not in non-HCC cells (L-02, SW1116, and Hela) (Fig. 1B,D). The GFP expression was vector dose-dependent (Fig. 1 C,E). However, as compared with the CMV promoter which is known to be one of the strongest promoters in a wide range of cell lines, the transcriptional activity of the AFP promoter was remarkably lower (Fig. 1E).


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)

Activity and tissue-specificity of the AFP promoter.(A) Construction of recombinant lentiviral vectors containing GFP reporter gene driven by AFP promoter or CMV promoter (AFP-GFP and CMV-GFP) for AFP promoter assay. (B,D) The AFP promoter is active and HCC tissue-specific. GFP was highly expressed in AFP-producing HCC cells (HepG2, Hep3B, and HCCLM3) but not in non-HCC cells (normal hepacyte L-02, cervical cancer cell Hela, and colon cancer cell SW1116). (C,E) The AFP promoter was efficient for transgenic expression but its activity was weaker than CMV-promoter, as exemplified by HCCLM3 cells infected with AFP-GFP (MOI = 5–80) and CMV-GFP (MOI = 5).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0053072-g001: Activity and tissue-specificity of the AFP promoter.(A) Construction of recombinant lentiviral vectors containing GFP reporter gene driven by AFP promoter or CMV promoter (AFP-GFP and CMV-GFP) for AFP promoter assay. (B,D) The AFP promoter is active and HCC tissue-specific. GFP was highly expressed in AFP-producing HCC cells (HepG2, Hep3B, and HCCLM3) but not in non-HCC cells (normal hepacyte L-02, cervical cancer cell Hela, and colon cancer cell SW1116). (C,E) The AFP promoter was efficient for transgenic expression but its activity was weaker than CMV-promoter, as exemplified by HCCLM3 cells infected with AFP-GFP (MOI = 5–80) and CMV-GFP (MOI = 5).
Mentions: A recombinant lentiviral vector containing GFP reporter gene driven by AFP promoter was constructed (AFP-GFP) (Fig. 1A). The activity and tissue-specificity of the AFP promoter were assayed by infecting the AFP-producing and non-AFP-producing cells with the AFP-GFP vector. GFP reporter assay showed that the AFP-promoter could efficiently drive GFP expression in AFP-producing HCC cells (HepG2, Hep3B, and HCCLM3) but not in non-HCC cells (L-02, SW1116, and Hela) (Fig. 1B,D). The GFP expression was vector dose-dependent (Fig. 1 C,E). However, as compared with the CMV promoter which is known to be one of the strongest promoters in a wide range of cell lines, the transcriptional activity of the AFP promoter was remarkably lower (Fig. 1E).

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