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.
Vigneswaran
Nadarajah
N
Department of Diagnostic Sciences, The University of Texas Health Science Center at Houston, Dental Branch, 6516, MD Anderson Blvd., Room 3.094G, Houston, Texas 77030, USA. Nadarajah.Vigneswaran@UTH.TMC.EDU
Wu
Jean
J
Nagaraj
Nagathihalli
N
James
Rohaizah
R
Zeeuwen
Patrick
P
Zacharias
Wolfgang
W
D002294:Carcinoma, Squamous Cell / Q000201:enzymology / Q000235:genetics* / Q000556:secondary; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D055332:Cystatin M; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009062:Mouth Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D009361:Neoplasm Invasiveness / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics*; D000099240:Asparaginyl Endopeptidase
D055332:Cystatin M; D015891:Cystatins; D003546:Cysteine Endopeptidases; D034741:RNA, Small Interfering; D000099240:Asparaginyl Endopeptidase; C525104:CST6 protein, human