Aims: Ovarian cancer is the leading cause of cancer death among gynaecological malignancies. Elevated expression of Rab25 has been seen in this malignancy. To better understand its role in maintaining the malignant phenotype, we used RNA interference (RNAi) directed against Rab25 in our study. RNAi provides a new, reliable method to investigate gene function and has the potential for gene therapy. The aim of the study was to examine the anti-tumour effects elicited by a decrease in the level of Rab25 by RNAi and its possible mechanism of action.
Methods: According to the Rab25 mRNA sequence in Genbank, a pair of 64 nt oligonucleotides were designed and synthesised, each containing the sites of restriction endonuclease at both ends. Oligonucleotides were annealed and ligated with linearised pSUPER by ligase. The recombinants (named pSUPER/Rab25 siRNA) were finally sequenced and identified by enzyme cutting and sequencing. The human ovarian cell line A2780 was grown without transfection, transfection with empty vector and with pSUPER/Rab25 siRNA with electroporation. The inhibitory effect was examined by RT-PCR, MTT, FCM and tumour growth of athymic nude mice.
Results: Rab25 siRNA expression vector was successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragment revealed the same sequence as synthesised siRNA oligonucleotides. Cells transfected with Rab25 siRNA can specifically knock down the transcription of Rab25, exhibiting cells with slower proliferation, increased apoptosis, and decreased tumour growth.
Conclusions: Rab25 siRNA expression vector has been successfully constructed, and it could inhibit the tumour growth both in vitro and in vivo. Our data suggest that the Rab25 signalling pathway plays a role in the regulation of cell proliferation and apoptosis in ovarian cancer cells, which indicates that the Rab25 gene plays a definite role in the development and aggressiveness of human ovarian cancer and should be further elucidated as a possible therapeutic target of ovarian cancer.