Construction and identification of recombinant RNA interference plasmid targeting to STAT5

Objective To construct and identify recombinant RNA interference plasmid targeting to STAT5.Methods Three pairs of DNA sequence containing small hairpin structure of specific siRNA targeting to STAT5A were designed and synthesized.The complement form obtained by annealing was inserted into vector Pgenesil-1 to construct the recombinant plasmid which was identified by enzyme digestion and DNA sequencing.The recombinant plasmid was transfected into human hepatocellular carcinoma cell line SMMC7721 by lipofectamineTM 2000 and the suppressive effect on STAT5 was assayed by semi-quantitive RT-PCR and western blot.Results The enzyme digestion analysis and DNA sequencing showed that RNA interference expression vector targeting to STAT5 was constructed successfully.The sequence-specific small hairpin RNA(shRNA) could effectively and specifically inhibit STAT5 gene expression at both mRNA and protein levels in SMMC7721 cell line.The inhibition rates of STAT5 mRNA expression were 70.43%,43.02% and 45.07% in SMMC7721-STAT5A1,SMMC7721-STAT5A2,SMMC7721-STAT5A respectively.The inhibition rates of STAT5 protein expression were 67.45%,37.36% and 41.86% in the 3 groups respectively.And Pgenesil-1-STAT5A1 had the most intense inhibitory effect.Conclusions RNA interference expression vector targeting to STAT5 was successfully constructed.This may lay a foundation for further study of cancer gene therapy in vivo using the RNA interference vector.