Construction And Functional Study Of SiRNA Mammalian Expression Vector Targeted To HER-3

Objective: The subject was to project and construct definite RNA interfere plasmid vector targeted to HER3 gene mRNA and transfected human breast cancer cell line MBA-MB-453, to investigate the interfere efficiency of HER3 in the cell lines and the effect on cell proliferation of knockdown HER3, and to explore the feasibility of siRNA technology in preventing and treating the breast cancer.Methods: Using the RNAi-Ready-pSIREN-RetroQ-TetP plasmid designed and constructed two specificity RNA interfere recombinant plasmid vectors that aimed at HER3 gene mRNA and had the inherited stably encoding hairpin construction, cotransfected HER3 positive human breast cancer MDA-MB-453 cell lines mediated by Lipofectin2000 and detected mRNA change with RT-PCR and protein expression with Western blotting technology and Flow cytometry. Measure cell proliferation activity with MTT chromatometry.Results: (1). DNA sequencing results displayed that two RNA interfere recombinant plasmids (named according to the respective site of shRNA sequence: RNAi-HER3-131 and RNAi-HER3-326) were accordance with experiment design. (2). By RT-PCR the mRNA expression level of HER3 decreased obviously in RNAi-HER3-131 group than that in control after 24 hours of transfection, but the mRNA expression level of HER3 in RNAi-HER3-326 group did not have obvious difference with that in control group after 24 hours of transfection. It suggested that RNAi-HER3-131 can significantly suppress HER3 expression. (3). The result of Western blotting and Flow cytometry indicated that the expression level of HER3 protein has significant difference between control group and RNAi-HER3-131group after 24 hours of transfection; RNAi-HER3-326 group did not have obvious difference with control group;β-actin expression had not significant difference among the 3 group, it proved that RNAi-HER3-131 can significantly suppress HER3 expression. (4). MTT assay showed that MDA-MB-453 cell survival was inhibited by transfection of recombinant plasmids: RNAi-HER3-131 transfection recombinant plasmid can suppress MDA-MB-453 cell survival significantly, and it had significant difference with the control group and RNAi-HER3-326 group. There were not obvious difference between the control group and RNAi-HER3-326 group.Conclusions: We successfully established a cell model that HER3 expression is suppressed almost completely by siRNA. RNAi-HER3-131 inhibits the survival of MDA-MB-453 cells in vitro.