Abstract:

Objective    To design three siRNAs targeting against murine VEGF by RNA interference (RNAi) technique and then insert them into the pSliencer4.1 CMV neoexpression vector to investigate the influence on the VEGF gene silencing and biological activity of murine colon carcinoma CT-26 cells, and to explore a novel gene therapy for colon cancer. Methods     ① Three sequences targeting against VEGF were inserted into the pSliencer4.1 CMV neo expression vector after being screened, designed and synthesized. The sequences were identified by restriction enzyme digestion and DNA sequencing.  ② Murine colon adenocarcinoma CT26 cells were divided into 6 groups: the recombinant plasmid V1, V2 and V3 groups(groups V1, V2 and V3), the Lipofectamine group(group Lp), the cell group(group c)and the scrambled group(group Nc). The recombinant vector was transferred into colon carcinoma CT-26 cells using Lipofectamine. Efficiencies of transcription and translation of VEGF were determined by semi-quantitative RT-PCR, Western blot and flow cytometry; and the cell proliferation， cell cycle distribution and cell apoptosis were measured by MTT and flow cytometry.  Results     ① The pSliencer4.1 CMV neo expression vector was successfully constructed and the sequence of the constructed recombinant plasmid was correctly identified by restriction enzyme digestion and DNA sequencing.   ②Expression of VEGF decreased markedly compared with the control groups(P<0.01)， and more markedly in groups V1 and V2(P<0.05, groups V1 and V2 vs group V3). ③  The growth of CT26 cells was significantly inhibited in groups V1,V2 and V3 compared with the control groups (P<0.01), and more significantly in groups V1 and V2 (P<0.05, groups V1 and V2 vs group V3). ④ Analysis of the cell cycle distribution showed that the ratio of cells at the G0/G1 phase increased and at S and G2/M phases decreased in groups V1 and V2 compared with group V3 and the control groups (P<0.05). Conclusion     ① The pSliencer4.1 CMV neo expression vector targeting against VEGF was successfully constructed. ②  The pSilencer4.1 CMV neoV1/ V2/ V3siRNA transfecting target CT-26 cells using Lipofectamine has a remarkable gene-silencing effect.

Key words: Vascular endothelial growth factor； Colon carcinoma； Proliferation； Gene silencing； RNA interference