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RNAi-mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion, angiogenesis and tumor growth in gliomas

Abstract

RNA interference (RNAi) provides a powerful method for gene silencing in eukaryotic cells, including proliferating mammalian cells. Here, we determined whether RNAi could be utilized to inhibit the expression of proteases implicated in the extracellular matrix degradation, which is characteristic of tumor progression. We have previously shown that antisense stable clones of uPAR and cathepsin B were less invasive and did not form tumors when injected intracranially ex vivo. Since antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high molar concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPAR and cathepsin B in this study. We found that the expression of double-stranded RNA leads to the efficient and specific inhibition of endogenous uPAR and cathepsin B protein expression in glioma cell lines as determined by Western blotting. We also found the RNAi of uPAR and cathepsin B reduces glioma cell invasion and angiogenesis in in vitro and in vivo models. Intratumoral injections of plasmid vectors expressing hpRNA for uPAR and cathepsin B resulted in the regression of pre-established intracranial tumors. Further, RNAi for uPAR and cathepsin B inhibited cell proliferation and reduced the levels of pERK and pFAK compared to controls. Taken together, our findings indicate for the first time that RNAi operates in human glioma cells with potential application for cancer gene therapy.

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Abbreviations

RNAi:

RNA interference

uPA:

urokinase-type plasminogen activator

uPAR:

uPA receptor

CMV:

cytomegalovirus

SV40:

simian virus type 40

PCR:

polymerase chain reaction

PBS:

phosphate-buffered saline

FITC:

fluoresceine-5-isothiocyanate

DiI:

1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanineperchlorate

DiO:

3,3'-dioctadecyloxacarbocyanine perchlorate

GFP:

green fluorescent protein

ECM:

extracellular matrix

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Acknowledgements

We would like to thank Noorjehan Ali for technical assistance, Tina Wilson for preparing the manuscript and Sushma Jasti and Diana Meister for manuscript review. Supported by National Cancer Institute Grants CA 85216, CA 75557 and CA 92393 (to JSR).

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Correspondence to Jasti S Rao.

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Gondi, C., Lakka, S., Dinh, D. et al. RNAi-mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion, angiogenesis and tumor growth in gliomas. Oncogene 23, 8486–8496 (2004). https://doi.org/10.1038/sj.onc.1207879

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