Construction and identification of Rab25 siRNA expression vector and its expression in oarian carcinoma cell line A2780

Objective To construct Rab25 siRNA expression vector in order to explore a new method of ovarian cancer gene therapy,and to assess its function on ovarian carcinoma cell A2780. Methods According to Rab25 mRNA sequence in the Genebank,a pair of 64-nt oligonucleotides,each containing the sites of restriction endonuclease at both ends,were designed and synthesized. Oligonucleotides were annealed and ligated with linearized pSUPER by T4DNA ligase. The recombinants (named pSUPER /Rab25 siRNA ) were finally sequenced and identified by enzyme cutting and sequencing. RT-PCR analysis was taken to show the change of Rab25 after the constructed plasmid had been transfeeted into A2780 cells. Results Rab25 siRNA expression vector was successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragment revealed the same sequence as synthesized siRNA oligonucleotides. The result of RT-PCR showed that Rab25 siRNA plasmid had inhibited Rab25 expression in A2780 cells obviously. Conclusion Rab25 siRNA expression vector has been successfully constructed,which will facilitate further studies of Rab25 function and its application in the treatment of ovarian cancer.