Construction of APE1 siRNA expression vector and its inhibition to APE1 in osteosarcoma cells / 第三军医大学学报

ABSTRACT

Objective To construct DNA damage and repair gene apurinic/apyrimidinic endonuclease (APE1) siRNA expression vector pSilence APE1 and investigate its inhibitory effect on the expression of APE1 in osteosarcoma cell HOS and 9901 in vitro. Methods An expression plasmid of a short hairpin RNA target APE1, pSilence APE1 was constructed and transfected to 9901 and HOS cell by lipofectamine. The expression of APE1 protein in HOS and 9901 osteosarcoma cells posttransfected with pSilence APE1 was detected by immunohistochemistry. Meanwhile, the dose-effect and time-effect relationship of APE1 gene silence induced by pSilence APE1 were measured using Western blot analysis. Results After evaluation and sequencing, the APE1 siRNA expression vector pSilence APE1 was constructed successfully. The result of Western blotting and immunohistochemistry showed that APE1 protein in osteosarcoma cells could be knocked down specifically by pSilence APE1, and the inhibition rate of APE1 expression was 72%-95%. The best inhibition of expression of APE1 gene was 3.0 ?g and at the 72 h using pSilence APE1. Conclusion APE1 siRNA expression vector pSilence APE1 is successfully constructed that can significantly knock down APE1 gene expression in osteosarcoma cells.