Silencing of cystatin M in metastatic oral cancer cell line MDA-686Ln by siRNA increases cysteine proteinases and legumain activities, cell proliferation and in vitro invasion

Cystatins are inhibitors of lysosomal cysteine proteinases. Cystatin M demonstrates more diverse tissue distribution, target specificity and biological function than other cystatins from the same family. We utilized small interference RNAs (siRNA) to silence cystatin M gene expression in a metastatic oral cancer cell line (MDA-686Ln) that expresses a high level of cystatin M. We tested four different siRNAs targeted to different sites of the cystatin M mRNA, and found three out of the four siRNAs were effective in suppressing cystatin M expression by > 50% at both mRNA and protein levels, as measured by quantitative real-time RT-PCR and Western blotting. We used siRNA-#1, which demonstrated highest efficiency of silencing cystatin M, to evaluate the phenotypic outcome of silencing cystatin M in MDA-686Ln cells. Cystatin M inhibition significantly increased the enzymatic activities of cathepsins B and L and legumain while reducing cysteine protease inhibitor activity both in the media and intracellularly. MDA-686Ln cells treated with siRNA#1 demonstrated markedly increased proliferation rate, in vitro motility and Matrigel invasiveness. Collectively, our data show that silencing of cystatin M in tumor cells not only increases their invasion and motility via cysteine–proteinase-dependent pathways, but also renders them hyperproliferative through a currently unknown mechanism.