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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

Efficacy of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi in vivo.(A,B) Mouse model of HCC via orthotopic implantation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intravenous injection of the AFP-Cre/LoxP-shRNA-Beclin1 did not knockdown Beclin 1 gene. GFP indicator analysis showed no GFP expression after the LoxP-shRNA vector was intravenously given, suggesting that the system did not enter the tumor in the liver. (C,D) Mouse model of HCC via subcutaneous inoculation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intratumoral injection of the AFP-Cre/LoxP-shRNA-Beclin1 could efficiently silence target gene (Beclin 1) in vivo. GFP indicator indicated that the AFP-Cre/LoxP-shRNA could efficiently infect and work in HCC tissue in vivo (GFP fluorescence diminished after intratumoral injection of the AFP-Cre/LoxP-shRNA, the LoxP-shRNA alone served as control). (Cre+LoxP: infection of AFP-Cre/LoxP-shRNA-Beclin1; AFP-Cre and LoxP-shRNA: infection of AFP-Cre or LoxP-shRNA alone).
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pone-0053072-g004: Efficacy of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi in vivo.(A,B) Mouse model of HCC via orthotopic implantation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intravenous injection of the AFP-Cre/LoxP-shRNA-Beclin1 did not knockdown Beclin 1 gene. GFP indicator analysis showed no GFP expression after the LoxP-shRNA vector was intravenously given, suggesting that the system did not enter the tumor in the liver. (C,D) Mouse model of HCC via subcutaneous inoculation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intratumoral injection of the AFP-Cre/LoxP-shRNA-Beclin1 could efficiently silence target gene (Beclin 1) in vivo. GFP indicator indicated that the AFP-Cre/LoxP-shRNA could efficiently infect and work in HCC tissue in vivo (GFP fluorescence diminished after intratumoral injection of the AFP-Cre/LoxP-shRNA, the LoxP-shRNA alone served as control). (Cre+LoxP: infection of AFP-Cre/LoxP-shRNA-Beclin1; AFP-Cre and LoxP-shRNA: infection of AFP-Cre or LoxP-shRNA alone).

Mentions: The AFP-Cre/LoxP-shRNA system was successfully established (Fig. 3A). The CMV-eGFP indicator inside could well indicate whether the system worked as desired (Fig. 3B,C). The GFP fluorescence diminished after AFP-Cre and LoxP-shRNA co-infected AFP-producing HCC cells (HCCLM3, HepG2, and Hep3B) while it did not change as AFP-Cre and LoxP-shRNA infected the non-HCC cells (L-02, Hela, and SW1116) (Fig. 3C). To assess the effectiveness and tissue-specificity of the AFP-Cre/LoxP-shRNA system, AFP-producing HCC cells (HCCLM3, HepG2, and Hep3B), normal hepatocyte (L-02), and non-HCC cancer cells (Hela and SW1116) were infected with the AFP-Cre/LoxP-shRNA system at a MOI of 1,5,10 or 20. Q-PCR and western blot analysis showed that the system could efficiently silence Beclin 1 gene in only AFP-producing HCC cells (Fig. 3C,D). It could effectively knockdown target gene at a MOI of 10–20 instead of the high titer required for the AFP-miRNA system (Fig. 3D). The effectiveness of the system was further examined in vivo. Systemic administration of the AFP-Cre/LoxP-shRNA system was firstlytested. Nude mouse model of HCC via orthotopic implantation was established using AFP-producing HCC cells (HCCLM3) and the AFP-Cre/LoxP-shRNA-Beclin1 was intravenously injected (Fig. 4B). Q-PCR and western blot analysis indicated that there was no significant decrease of Beclin 1 gene after the AFP-Cre/LoxP-shRNA-Beclin1 was given (Fig. 4A). Intravenous injection of LoxP-shRNA with CMV-eGFP indicator showed no GFP expression in HCC tumor, which suggested that no vectors were delivered into the tumor cells (Fig. 4B). The ineffectiveness of systemic administration was thought to be attributed to the disadvantages of the nude mouse model and lentivirus-based vector. The effectiveness of in vivo application of the system was then examined by intratumorally administration. Nude mouse model of HCC via subcutaneous injection was established using HCCLM3 cells (Fig. 4D). The AFP-Cre/LoxP-shRNA-Beclin1 was intratumorally injected into the subcutaneous tumor. The GFP indicator analysis showed that the system could efficiently infect HCC cells (Fig. 4D). Q-PCR and western blot analysis showed that it could efficiently knockdown the Beclin 1 gene of the HCCLM3 tumor tissues in vivo (Fig. 4C).


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)

Efficacy of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi in vivo.(A,B) Mouse model of HCC via orthotopic implantation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intravenous injection of the AFP-Cre/LoxP-shRNA-Beclin1 did not knockdown Beclin 1 gene. GFP indicator analysis showed no GFP expression after the LoxP-shRNA vector was intravenously given, suggesting that the system did not enter the tumor in the liver. (C,D) Mouse model of HCC via subcutaneous inoculation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intratumoral injection of the AFP-Cre/LoxP-shRNA-Beclin1 could efficiently silence target gene (Beclin 1) in vivo. GFP indicator indicated that the AFP-Cre/LoxP-shRNA could efficiently infect and work in HCC tissue in vivo (GFP fluorescence diminished after intratumoral injection of the AFP-Cre/LoxP-shRNA, the LoxP-shRNA alone served as control). (Cre+LoxP: infection of AFP-Cre/LoxP-shRNA-Beclin1; AFP-Cre and LoxP-shRNA: infection of AFP-Cre or LoxP-shRNA alone).
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Related In: Results  -  Collection

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

pone-0053072-g004: Efficacy of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi in vivo.(A,B) Mouse model of HCC via orthotopic implantation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intravenous injection of the AFP-Cre/LoxP-shRNA-Beclin1 did not knockdown Beclin 1 gene. GFP indicator analysis showed no GFP expression after the LoxP-shRNA vector was intravenously given, suggesting that the system did not enter the tumor in the liver. (C,D) Mouse model of HCC via subcutaneous inoculation of HCCLM3 cells was established. Q-PCR and western blot analysis showed that intratumoral injection of the AFP-Cre/LoxP-shRNA-Beclin1 could efficiently silence target gene (Beclin 1) in vivo. GFP indicator indicated that the AFP-Cre/LoxP-shRNA could efficiently infect and work in HCC tissue in vivo (GFP fluorescence diminished after intratumoral injection of the AFP-Cre/LoxP-shRNA, the LoxP-shRNA alone served as control). (Cre+LoxP: infection of AFP-Cre/LoxP-shRNA-Beclin1; AFP-Cre and LoxP-shRNA: infection of AFP-Cre or LoxP-shRNA alone).
Mentions: The AFP-Cre/LoxP-shRNA system was successfully established (Fig. 3A). The CMV-eGFP indicator inside could well indicate whether the system worked as desired (Fig. 3B,C). The GFP fluorescence diminished after AFP-Cre and LoxP-shRNA co-infected AFP-producing HCC cells (HCCLM3, HepG2, and Hep3B) while it did not change as AFP-Cre and LoxP-shRNA infected the non-HCC cells (L-02, Hela, and SW1116) (Fig. 3C). To assess the effectiveness and tissue-specificity of the AFP-Cre/LoxP-shRNA system, AFP-producing HCC cells (HCCLM3, HepG2, and Hep3B), normal hepatocyte (L-02), and non-HCC cancer cells (Hela and SW1116) were infected with the AFP-Cre/LoxP-shRNA system at a MOI of 1,5,10 or 20. Q-PCR and western blot analysis showed that the system could efficiently silence Beclin 1 gene in only AFP-producing HCC cells (Fig. 3C,D). It could effectively knockdown target gene at a MOI of 10–20 instead of the high titer required for the AFP-miRNA system (Fig. 3D). The effectiveness of the system was further examined in vivo. Systemic administration of the AFP-Cre/LoxP-shRNA system was firstlytested. Nude mouse model of HCC via orthotopic implantation was established using AFP-producing HCC cells (HCCLM3) and the AFP-Cre/LoxP-shRNA-Beclin1 was intravenously injected (Fig. 4B). Q-PCR and western blot analysis indicated that there was no significant decrease of Beclin 1 gene after the AFP-Cre/LoxP-shRNA-Beclin1 was given (Fig. 4A). Intravenous injection of LoxP-shRNA with CMV-eGFP indicator showed no GFP expression in HCC tumor, which suggested that no vectors were delivered into the tumor cells (Fig. 4B). The ineffectiveness of systemic administration was thought to be attributed to the disadvantages of the nude mouse model and lentivirus-based vector. The effectiveness of in vivo application of the system was then examined by intratumorally administration. Nude mouse model of HCC via subcutaneous injection was established using HCCLM3 cells (Fig. 4D). The AFP-Cre/LoxP-shRNA-Beclin1 was intratumorally injected into the subcutaneous tumor. The GFP indicator analysis showed that the system could efficiently infect HCC cells (Fig. 4D). Q-PCR and western blot analysis showed that it could efficiently knockdown the Beclin 1 gene of the HCCLM3 tumor tissues in vivo (Fig. 4C).

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