Effects Of Silencing Rabex-5Expression By Lentiviral-mediated RNA Interference On The Biological Behavior Of Breast Cancer Cell Lines

Objective：To construct a lentiviral vector for RNA interference(RNAi) of Rabex-5gene and to detect its silence effect on Rabex-5inMCF-7cells and its impacts on biological behaviors after interference．Theaim of the present study is to explore the prospects of lentivirus-mediatedsilencing Rabex-5application in gene therapy for breast cancer in order toprovide a new treatment for breast cancer.Methods：The synthesised single-stranded primer was annealed todouble-stranded oligo sequences and sub-cloned into linear pMAGiclentiviral plasmid vector digested by enzyme Age I and EcoR I. DH5αcompetent cells were transformed and the positive clone were screened,identified and sequenced. The recombinant lentivirus was packaged intothe mature lentivirus by293FT cells and used to infect MCF-7cells. Theexpression of Rabex-5in the cells was detected by real-time PCR andWestern blot. The effects of recombinant lentivirus on cell proliferation,invasion and migration ability were analyzed by CCK-8method andTranswell chamber test． Results：PCR and sequencing verified that the recombinant lentivirusplasmid Rabex5-shRNA was successfully constructed. Compared with thenegative control and the blank control cells, Rabex-5mRNA and proteinlevel were reduced in MCF-7cells infected by lentivirus. RNAi3had thebest effect of gene silence. The downregulation of Rabex-5expressioncould inhibit invasion and migration ability of MCF-7cells (P＜0.05)，while cell proliferation was not obviously affected (P＞0.05).Conclusion：The lentiviral RNAi expression vector targeting Rabex-5gene is successfully constructed and it can downregulate the expression ofRabex-5in MCF-7cells. Our research shows that Rabex-5plays animportant role in invasion and migration of breast cancer, and providesexperimental evidences for Rabex-5-mediated gene therapy against breastcancer.