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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 of AFP-miRNA system for HCC tissue-specific RNAi.(A) Construction of recombinant lentiviral vectors contained miRNA targeting Beclin 1 gene driven by AFP promoter or CMV promoter (AFP-miRNA and CMV-miRNA). (B) The AFP-miRNA system could efficiently infected HCC cells (more than 99%), as exemplified by HCCLM3 cells. (C) Q-PCR and western blot analysis showed that the AFP-miRNA system could downregulate target gene (Beclin 1) of HCC cells but required high titer (MOI>80), as compared with positive control CMV-miRNA. (D) Cell viability assay showed that the high titer required for effective AFP-miRNA-mediated RNAi was cytotoxic to target HCC cells, as exemplified by HCCLM3 and HepG2 cells.
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pone-0053072-g002: Effectiveness of AFP-miRNA system for HCC tissue-specific RNAi.(A) Construction of recombinant lentiviral vectors contained miRNA targeting Beclin 1 gene driven by AFP promoter or CMV promoter (AFP-miRNA and CMV-miRNA). (B) The AFP-miRNA system could efficiently infected HCC cells (more than 99%), as exemplified by HCCLM3 cells. (C) Q-PCR and western blot analysis showed that the AFP-miRNA system could downregulate target gene (Beclin 1) of HCC cells but required high titer (MOI>80), as compared with positive control CMV-miRNA. (D) Cell viability assay showed that the high titer required for effective AFP-miRNA-mediated RNAi was cytotoxic to target HCC cells, as exemplified by HCCLM3 and HepG2 cells.

Mentions: We first investigated the strategy of AFP-promoter driven miRNA for HCC-specific gene silencing. An AFP-miRNA lentiviral vector (AFP-miRNA) targeting Beclin 1 gene was constructed (Fig. 2A). A CMV promoter driven miRNA vector (CMV-miRNA) served as positive control (Fig. 2A). To assess the effectiveness of the AFP-miRNA, AFP-producing HCC cells (HCCLM3, HepG2, and Hep3B) were infected with the AFP-miRNA at various MOI. The AFP-miRNA was efficient in infecting all cell lines in vitro (Fig. 2B). Q-PCR and western blot analysis of Beclin 1 gene silencing showed that the AFP-miRNA could knockdown Beclin 1 but required considerably high titer (Fig. 2C). A MOI of more than 80 was required to achieve efficient gene silencing (Fig. 2C). As compared with positive control CMV-miRNA, the efficacy of the AFP-miRNA was remarkably weaker (Fig. 2C). The CMV-miRNA infection at a MOI of 20 could achieve silencing level equal to that of the AFP-miRNA infection at a MOI of 320 in AFP-producing HCC cells (Fig. 2C). As high titer was needed for effective gene silencing, the cytotoxicity of the AFP-miRNA against target cells was determined. Cell viability assay showed that the AFP-miRNA infection at required titer (MOI>80) led to considerable cell death. With the increase of MOI, the cytotoxicity significantly enhanced (Fig. 2D).


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 of AFP-miRNA system for HCC tissue-specific RNAi.(A) Construction of recombinant lentiviral vectors contained miRNA targeting Beclin 1 gene driven by AFP promoter or CMV promoter (AFP-miRNA and CMV-miRNA). (B) The AFP-miRNA system could efficiently infected HCC cells (more than 99%), as exemplified by HCCLM3 cells. (C) Q-PCR and western blot analysis showed that the AFP-miRNA system could downregulate target gene (Beclin 1) of HCC cells but required high titer (MOI>80), as compared with positive control CMV-miRNA. (D) Cell viability assay showed that the high titer required for effective AFP-miRNA-mediated RNAi was cytotoxic to target HCC cells, as exemplified by HCCLM3 and HepG2 cells.
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Related In: Results  -  Collection

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

pone-0053072-g002: Effectiveness of AFP-miRNA system for HCC tissue-specific RNAi.(A) Construction of recombinant lentiviral vectors contained miRNA targeting Beclin 1 gene driven by AFP promoter or CMV promoter (AFP-miRNA and CMV-miRNA). (B) The AFP-miRNA system could efficiently infected HCC cells (more than 99%), as exemplified by HCCLM3 cells. (C) Q-PCR and western blot analysis showed that the AFP-miRNA system could downregulate target gene (Beclin 1) of HCC cells but required high titer (MOI>80), as compared with positive control CMV-miRNA. (D) Cell viability assay showed that the high titer required for effective AFP-miRNA-mediated RNAi was cytotoxic to target HCC cells, as exemplified by HCCLM3 and HepG2 cells.
Mentions: We first investigated the strategy of AFP-promoter driven miRNA for HCC-specific gene silencing. An AFP-miRNA lentiviral vector (AFP-miRNA) targeting Beclin 1 gene was constructed (Fig. 2A). A CMV promoter driven miRNA vector (CMV-miRNA) served as positive control (Fig. 2A). To assess the effectiveness of the AFP-miRNA, AFP-producing HCC cells (HCCLM3, HepG2, and Hep3B) were infected with the AFP-miRNA at various MOI. The AFP-miRNA was efficient in infecting all cell lines in vitro (Fig. 2B). Q-PCR and western blot analysis of Beclin 1 gene silencing showed that the AFP-miRNA could knockdown Beclin 1 but required considerably high titer (Fig. 2C). A MOI of more than 80 was required to achieve efficient gene silencing (Fig. 2C). As compared with positive control CMV-miRNA, the efficacy of the AFP-miRNA was remarkably weaker (Fig. 2C). The CMV-miRNA infection at a MOI of 20 could achieve silencing level equal to that of the AFP-miRNA infection at a MOI of 320 in AFP-producing HCC cells (Fig. 2C). As high titer was needed for effective gene silencing, the cytotoxicity of the AFP-miRNA against target cells was determined. Cell viability assay showed that the AFP-miRNA infection at required titer (MOI>80) led to considerable cell death. With the increase of MOI, the cytotoxicity significantly enhanced (Fig. 2D).

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