Objective: To construct an RNA interference vector to down-regulate X-linked inhibitor of apoptosis (XIAP) gene and study the RNA interference effect on the cell cycle and growth of ovarian cancer.
Methods: Oligonucleotides of 64 base pairs for hairpin RNA targeting XIAP were designed, chemically synthesized, annealed, and cloned into the pSUPER vector. After identification by restriction digestion, the correct vectors were transiently transfected into SKOV3 cells, a human ovarian cancer cell line. The XIAP mRNA was detected by RT-PCR. The proteins were detected by western blot and indirect immunofluorescence staining. Flow cytometry (FCM) analysis and methyl thiazolyl tetrazolium (MTT) assay method were applied to measure cell cycle, cell growth and sensitiveness to cisplatin.
Results: SKOV3 cells had a high level expression of XIAP. The vector of RNA interference, which can interfere with XIAP gene was successfully constructed. After transient transfection, the expression of XIAP protein was significantly decreased in SKOV3 cells and the value of relative density was 3584 +/- 124, 2138 +/- 65, 1973 +/- 80 and 110 +/- 12, respectively (P = 0.0334). At the same time, the expression of XIAP mRNA was decreased accordingly and the value of relative density was 6674 +/- 274, 4532 +/- 107, 2322 +/- 57 and 1864 +/- 78, respectively (P = 0.0127). The FCM results showed that, the vector could increase the number of cells in G(1) phase compared with parent cells and compared with the cells transfected with pSUPER (P < 0.05); the number in S phase decreased compared with parent cells and compared with the cells transfected with pSUPER (P < 0.05). MTT results showed that pSUPER-siXIAP could inhibit the growth and proliferation of SKOV3 cells in a time-dependent manner, and inhibit the tumor cell growth.
Conclusions: We successfully constructed an expressing hairpin RNA against the XIAP vector. The vector can effectively silence XIAP gene, increase the number of cells in G(1) phase, and slow down the growth of tumor cells. This may be a useful therapeutic strategy for XIAP over-expressing ovarian carcinomas.