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

Nadarajah Vigneswaran, Jean Wu, Nagaraj Nagathihalli, Rohaizah James, Patrick Zeeuwen, Wolfgang Zacharias

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)898-907
Number of pages10
JournalLife Sciences
Volume78
Issue number8
DOIs
StatePublished - Jan 18 2006
Externally publishedYes

Fingerprint

asparaginylendopeptidase
Cystatin M
Cysteine Proteases
Mouth Neoplasms
Cell proliferation
RNA Interference
Cells
Cell Proliferation
RNA
Cell Line
Cystatins
Cysteine Proteinase Inhibitors
Cathepsin L
In Vitro Techniques
Cathepsin B
Messenger RNA
Tissue Distribution

Keywords

  • Cathepsins
  • Cystatin M
  • Legumain
  • Oral cancer
  • siRNA

ASJC Scopus subject areas

  • Pharmacology

Cite this

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. / Vigneswaran, Nadarajah; Wu, Jean; Nagathihalli, Nagaraj; James, Rohaizah; Zeeuwen, Patrick; Zacharias, Wolfgang.

In: Life Sciences, Vol. 78, No. 8, 18.01.2006, p. 898-907.

Research output: Contribution to journalArticle

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abstract = "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.",
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