Background: Gene targeting using short interfering RNA(siRNA) has become a common strategy to explore gene function because of its prominent efficacy and specificity. It is proven that the application of siRNA technology to gene therapy is effective. In this study, we constructed a siRNA expression plasmid against gene X-linked inhibitor of apoptosis (XIAP), and then used breast cancer cells MCF-7 to assess its functions.
Materials and methods: XIAP siRNA plasmid was constructed using an U6pro vector contained U6 promoter, After the plasmid had been transfected into MCF-7 cells and effected on the cell cycle, the expression change of XIAP was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The apoptosis of the transfected cells was analyzed by flow cytometry, and TUNEL method. The in vitro cellular growth activities were assayed by MTT incorporation. Twenty-four nude mice were randomly divided into 3 equal groups and were inoculated with electroinjection of blank plasmid, scrambled nucleotide control (control siRNA), or siRNA against XIAP subcutaneously respectively, then the appearance and size of tumors were observed. Four weeks later the mice were killed and the volumes of tumor were calculated so as to evaluate the therapeutic effects of siRNA against XIAP.
Results: The successful construction of siRNA against XIAP plasmid was identified with sequencing. After the siRNA expression vector was transfected into the MCF-7 cells, the expression of XIAP gene was inhibited significantly (by 90%). The cellular growth activities in the MCF-7 cells transfected with siRNA against XIAP plasmid decreased obviously. The siRNA against XIAP plasmid knocked down XIAP expression in MCF-7 cells obviously, arrested the cell cycle in G1 phase, inhibited cell proliferation significantly, and promoted cell apoptosis in a tendency. TUNEL assay and flow cytometry showed that the classic apoptosis characters of the MCF-7 cells transfected with siRNA against XIAP plasmid manifested an apoptosis rate of 77.2%, significantly higher than those in the control siRNA group and in the blank plasmid group (both p < 0.01). The growth speed and formation rate of xenograft tumor in mice transfected with siRNA against XIAP transfected mice slowed down significantly. By HE staining, a lot of necrotic tissues could be observed in the siRNA against XIAP transfected group, however, there was no similar inhibitive effect in the control siRNA or blank plasmid group.
Conclusion: This study represents that MCF-7 transfected cells with siRNA against XIAP remarkably suppress tumor growth and induces apoptosis, both in vitro and in vivo. This novel modality may be a promising tool for cancer therapy.