DNA Polymerases As Targets For Gene Therapy Of Hepatocyte Carcinoma

Background:Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with high mortality. Nowadays, treatments for HCC are still limited. Gene therapy, an ideal approach for its specific expression of exogenous genes in HCC cells driven by tissue-specific promoter, is a novel strategy for HCC therapy. However strategies based on correction of mutations or altered expression of genes responsible for the development/progression of HCC have limitations because these aberrant molecules are not presented in all cancerous cells. In the current work, we adopted a novel strategy by targeting the DNA replication step which is essential for proliferation of every cancer cell.Methods:A recombinant adenovirus was constructed with alpha fetoprotein (AFP) promoter-controlled expressions of artificial microRNAs targeting DNA polymerases α,δ, ε and recombinant active Caspase 3, namely Ad/AFP-Casp-AFP-amiR. And a recombinant adenovirus with AFP promoter only, namely Ad/AFP, was constructed as control.Results:In vitro:HepG2, Hep3B, and HL7702 cells were infected by two adenoviruses with MOI 50 respectively. Ad/AFP-Casp-AFP-amiR could efficiently inhibit the expression of the target polymerases in AFP-positive HCC cells at both RNA and protein levels and induced significant GO/1 phase arrest. The proliferation of HCC cells were significantly inhibited by Ad/AFP-Casp-AFP-amiR with increased apoptosis. On the contrary, the recombinant adenovirus Ad/AFP-Casp-AFP-amiR did not inhibit the expression of DNA polymerases α,δ or ε in AFP-negative human normal liver cell HL7702, and showed no effect on the cell cycle progression, proliferation or apoptosis. In vivo: Ad/AFP-Casp-AFP-amiR showed no significant inhibition on tumor growth but induced apoptosis in Hep3B xenograft in nude mice.Conclusion:Inhibition of DNA polymerases α,δ and ε by AFP promoter-driven artificial microRNAs can lead to effective growth arrest of AFP-positive HCC cells, which may represent a new approach for gene therapy by targeting genes which is essential for the growth/proliferation of cancer cells, avoiding the limitations set by any of the individually altered gene.