A novel HBV antisense RNA gene delivery system targeting hepatocellular carcinoma

Figure 1 Physical map of pEBAF-as-preS2. hepatocarcinoma specific preS2 antisense RNA expression vector, pEBAF-as-preS2 was constructed by inserting HBV preS2 gene (3203-3340) reversely downward the AFP promoter and enhancer (AFP-P/E) of plasmid pEBAF which contains the key element of EB virus duplication (Ori P and EBNA-1).

Figure 2 Construction of AFP-enhancing 4-element complex. 0.2 μg plasmid DNA was independently mixed dropwise with various quantity of polypeptide conjugation for 30 min at room temperature, and then the mixture was analyzed with 1% agrose gel electrophorosis. Fig 2 showed the DNA retardation of different complex mixed with DNA and peptide in differ-ent ratios. The ratio of DNA and peptide in Lane 1 to 7 is 0, 1/ 1,1/2. 1/2.5, 1/3, 1/3.5. 1/4. M indicates DNA marker.

Figure 3 RT-PCR of tissues in nude mice treated AFP-enhanc-ing 4-element complex. Total RNAs of different tissues of Nude mice bearing hepatocarcinoma were extracted 3 days after treatmemt with AFP-enhancing 4-element complex and then RT-PCR was performed; the expression of HBV preS2 gene was analyzed. A. RT-PCR of beta-actin. (bands 1 to 7 stand for the PCR products of spermaduct, stomach, liver, spleen, heart, and tumors, M stands for the DNA marker); B. RT-PCR of HBV preS2 gene.

Figure 4 Effect of AFP-enhancing 4-element complex on HBV gene expression in HepG2. 2.15 cells.