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University Department of Surgery, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom [W. G. J., D. G., J. L., T. A. M., R. E. M.], and Department of Neurology, Kennedy Krieger Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 [R. A., J. L.]
Purpose: Hepatocyte growth factor/scatter factor (HGF/SF), via its receptor c-MET, has been implicated to play a pivotal role in breast cancer development and progression. This study examined a transgene—consisting of a combination of U1snRNA, hammerhead ribozyme, and antisense, designed to inhibit c-met expression—and its impact on the migration and in vitro invasion of breast cancer cells.
Experimental Design: A hammerhead ribozyme targeting human c-MET was cloned into a modified pZeoU1EcoSpe vector and transfected into breast cancer cells MDA MB 231 and MCF-7 by electroporation. Expression of MET mRNA and protein was determined. Migration and in vitro invasiveness of transfected cells were also analyzed.
Results: Breast cancer cells were transfected with the ribozyme-containing plasmids. Stable transfectants manifested an almost complete loss of MET mRNA and protein, as shown by reverse transcription-PCR, Northern blotting, and Western blotting, respectively, whereas the wild-type plasmid had no effects. Met-ribozyme transfected cells exhibited reduced migration and in vitro invasiveness through extracellular matrix (Matrigel), compared with the wild-type cells and cells transfected with empty plasmid.
Conclusions: These data show that targeting c-MET by way of a hammerhead ribozyme encoding antisense to c-MET is an effective approach in reducing the invasiveness of breast cancer cells.
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