Silencing Proteases in Glioma Figure 6. Involvement of a9b1 integrin on the migration potential of xenograft cells and the effect of bicistronic constructs on the glioma xenograft cell proliferation. (A) RT-PCR of 5310 and 4910 cells transfected with uPAR (U-fl), MMP-9 (M-fl), and cathepsin B (C-fl) expressing plasmid DNAs was performed as per standard protocols. Prominent increase in respective mRNA expressions noticed after U-fl, M-fl, and C-fl treatments. Further, quantification of the RT-PCR data (B) revealed significant increases in uPAR mRNA and MMP-9 mRNA expression after treatments with M-fl and C-fl, respectively in both the xenograft cells. (C) Wound healing indicative of increased migration potential noticed after treatments with U-fl, M-fl, and C- fl in 5310 cells was reduced with the same treatments in presence of a9b1 antibody. Photographs are the representative images obtained from three independent experiments. (D) Quantification of wound healing assay. Percent wound repair was calculated from the mean of the average width of the wound obtained from 3 independent experiments. Error bars indicate SEM. *p,0.05 vs. control. #p,0.05. (E) FACS analysis showing reduced a9b1 integrin levels in 4910 cells after MMP-9/uPAR (MU-sh) and MMP-9/cathepsin B (MC-sh) treatments. (F) Clonogeneic assay depicting the reduced proliferation of 4910 and 5310 cells after MU-sh and MC-sh treatments. After 14 days of incubation, the colonies containing more than 50 cells were counted. Images shown are the representatives obtained from three independent experiments. (G) MTT assay showing significant reduction in the proliferation of 4910 cells from Day 4 to Day 6 after MU-sh and MC-sh treatments. n = 3. *p,0.05 vs. control. doi:10.1371/journal.pone.0011583.g006 that has been implicated to play a key role in integrin-mediated signal transduction pathways [5,45]. The most important transmembrane receptors associated with uPAR signaling are the integrin family of ECM receptors [34]. uPAR/b1 integrin interactions are frequently associated with the activation of FAK and ERK, whereas uPAR/b3 integrin interactions are associated with the activation of Rac [20,46–48]. Prominent reduction in FAK mRNA levels with MU-sh and MC-sh treatments in both the xenograft cell lines in the present study demonstrated the involvement of integrins via FAK in inhibiting their adhesion and migration potential. Even though the migration potential of both the xenografts was significantly reduced with all the shRNA treatments, they behaved differently in regulating the mRNA expressions of various genes associated with migration. Reduced FAK, phospho-FAK, cSrc, phospho-Src, Arp2, F-actin, paxillin, phospho-paxillin protein levels after treatment with bicistronic constructs in 5310 cells further strengthened our hypothesis that our treatments inhibit the migratory potential of these xenografts. Taken together, our findings clearly demonstrated that shRNA- mediated silencing of MMP-9, uPAR, and cathepsin B inhibited adhesive, migrating and invasive potentials of glioma xenografts. Bicistronic constructs that can simultaneously downregulate MMP-9 and uPAR or MMP-9 and cathepsin B were more effective than any of the single constructs. We also show the involvement of several integrins, FAK and associated molecules in the effects mediated by our shRNA constructs. Furthermore, for PLoS ONE | www.plosone.org 9 July 2010 | Volume 5 | Issue 7 | e11583