Objective: To construct eukaryotic expression vectors by using the pSilencer3. 1-H1 neo vector for inhibiting human survivin gene by RNA interference, and to detect the effect of the silenced survivin gene on PC-3 cells.
Methods: Three target gene segments were synthesized and cloned into the pSilencer3. 1-H1 neo vector respectively to construct three recombinant eukaryotic expression vectors: pSilencer3. 1-SVV1, pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3, which were identified by enzyme digestion analysis and DNA sequencing. Then the PC-3 cells were transfected with the recombinant vectors and the interference effect detected by RT-PCR, Western blot and immunohistochemical staining. The apoptosis index of the PC-3 cells was detected by flow cytometry and their proliferation detected by MTT method.
Results: Enzyme digestion analysis and DNA sequencing showed that three target segments were cloned into pSilencer3. 1-H1-neo vectors. The results of RT-PCR, Western blot and immunohistochemical staining indicated that pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors could knock down the transcription and expression of survivin gene. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased by 10% - 15% and their growth obviously slowly down.
Conclusion: The transcription and expression of survivin gene were inhibited effectively by the recombinant eukaryotic expression vectors (pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3) in the prostate cancer cell line PC-3. After transfected with pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased and their growth inhibited.