Targeted Activation Of Apoptosis Of Hepatocellular Cancer Cells By A Fusion Protein With A Human Scfv Antibody Mediated Survivin Specific SiRNA Delivery

RNA interference is one of the ideal targeted therapies at oncotherapy. However, before small interfering RNA (siRNA) can be used in the clinic, it is essential to develop a system to target their delivery. Antibody-mediated delivery is a new approach for targeting siRNA to appropriate cells. In this report, we designed an antibody-truncated protamine (tP) fusion protein to delivery siRNA to human hepatocarcinoma cells. The fusion protein, ScFv-tP, was constructed to contain a single chain of the human variable fragment, scFv, against one of the hepatocellular cancer cells surface antigen, and a truncated protamine (tP), which has the DNA binding activity. ScFv-tP was developed to provide targeted delivery of the siRNA into the hepatocellular cancer cells. Survivin was one of the IAP family proteins. It is said that Survivin was highly expressed in hepatocellular carcinoma tissues. Thus, Survivin specific-siRNA with fluorescein was specifically delivered into hepatocellular cancer cells using the ScFv-tP fusion protein, and effectively inhibited the expression of Survivin in vivo and in vitro, which leads to the apoptosis of hepatocellular cancer cells. Our results demonstrate a potential method for systemic, cell-type specific, scFv antibody mediated siRNA delivery.Part 1AIM: Construction, Expression, and Purification of scFv -tP Fusion protein.METHODS: The ScFv–tP fusion sequence was amplified from 2 PCR reactions, with the truncated human protamine 1 encoding sequence designed in the 3'ScFv primers. Sequences encoding scFv -tP were cloned into the pET32a and the fusion proteins were expressed in Escherichia coli BL21 and purified by Ni2+-NTA agarose according to the manufacturer's protocol. SDS-PAGE analysis was used for the detection of the products.RESULTS and CONCLUSION: Restriction endonuclease digestion analysis and DNA sequencing proved that the amplified ScFv sequence was correct, and the plasmids of the ScFv/tP fusion protein were successfully constructed. SDS-PAGE analysis showed that the fusion proteins were successfully expressed and purified after induced in E.coli BL21 by IPTG. The ScFv/tP fusion protein was at 42kDa tested by SDS-PAGE analysis.Part 2AIM:To test the ability of the ScFv/tP fusion protein.METHODS: The HepG2 cells was chosen to test the ability of the ScFv/tP fusion protein compared to the 7701 cells. DNA binding ability was verified by Gel shift assay, and the internalization function was confirmed by indirect immunofluorescence staining. Delivery of siRNA with fluorescence was assessed by the light and fluorescence microscope.RESULTS: Gel shift assay assured that the ScFv/tP fusion protein containing tP had DNA binding activity. Indirect immunofluorescence staining revealed that the ScFv/tP fusion protein was able to internalize into HepG2 cells but not 7701 cells. The observation by the light and fluorescence microscope showed that the ScFv/tP fusion protein could successfully deliver the siRNA with fluorescence into HepG2 cells but not 7701 cells.CONCLUSION: The ScFv/tP fusion protein obtained DNA binding activities and was able to internalize into HepG2 cells successfully. It could specifically deliver siRNA into HepG2 cells.Part 3AIM:To assess the apoptosis induced by the Survivin siRNA using the ScFv/tP fusion protein as a transfection reagent. METHODS: The HepG2 cells was chosen to assess the apoptosis by the ScFv/tP fusion protein compared to the 7701 cells by using RT-PCR, REAL-PCR,Western-Blot, Flow Cytometry, Tunel, MTT, trypan blue staining, PCR Array, Electron Microscopy, nude mice test and indirect immunofluorescence staining.RESULTS: After the transfection of Survivin siRNA by the ScFv/tP fusion protein, the apoptosis in the HepG2 cells was increasing significantly compared to the 7701 cells. The apoptosis in vivo was also increasing by using the the ScFv/tP fusion protein.CONCLUSION: The ScFv/tP fusion protein can be thought as a transfection reagent, it could specifically deliver siRNA into human hepatocellular cancer cells, such as HepG2 cells. The present studies may provide a theoretical foundation for the application of this ScFv in targeted delivery of DNA or siRNA to hepatocellular cancer cells.