Construction and identification of RNA interference adenovirus expression vector of targeting STAT3

Objective To construct RNA interference (RNAi) expression vector of targeting STAT3 and test the silencing effect by transfecting rat glioma cell to make foundation for exploring new way of gene therapy for cancer. Methods Two DNA sequences containing small hairpin structure were designed and synthesized. The complement form obtained by annealing was inserted into PGEM-T vector to construct the recombinant, the plasmid of which identified by enzyme digestion and DNA sequence analysis, was cloned into shuttle plasmid PDC316 and transfected into human embryonic kidney cells HEK293 by lipofectamine 2000 mediation, together with adenovirus-packaging plasmid pBHGE3. Based on the homologous recombination of the two plasmids within HEK293 cells, recombinant adenovirus (rAD)-STAT3 created was identified by PCR. rAD was purified using repeated plaque passages, proliferated using freezing and melting within HEK293 cells, and titrated using 50% tissue culture infective dose (TCID50) assay. The rAD expression vector was transfected into rat glioma to assay the gene and protein expressions of STAT3 by RT-PCR and Western-blotting. Negative plasmid transfected into the same cell line was as control group. Results The enzyme digestion analysis and DNA sequencing showed that RNAi expression vector of targeting STAT3 was constructed successfully. The expressions of mRNA and protein expressions of STAT3 in C6 cell transfected were weaker than that of control group. Conclusions RNAi expression vector of targeting STAT3 is successfully constructed and can inhibit STAT3 gene expression in C6 cell. It makes a foundation for gene therapy for cancer by RNAi technique to silence expression of gene STAT3 in cancer cells and induce apoptosis or inhibit proliferation of cancer cells.