Figure 4. MT1-MMP represses let-7 microRNA in vivo.
A. Full-length MT1-MMP and ΔC mutant were subcloned into pRetroX-Tight-Pur vector (Clontech, Mountainview, CA). Panc1 cells inducibly expressing MT1-MMP were then generated as previously described (Dangi-Garimella and Redig et al., 2010) to create Panc1-tet-V, Panc1-tet-MT and Panc1-tet-ΔC. These cells were grown on plastic, induced with doxycycline (2 μg/ml) for 24 hours, and then immunoblotted for MT1-MMP and α-tubulin (loading control) (upper panel). To examine MT1-MMP activity, the conditioned medium was analyzed for MMP-2 activation using gelatin zymography (lower panel). Data are representative of at least 4 separate experiments. B. Panc1-tet-V, Panc1-tet-MT and Panc1-tet-ΔC cells were grown on tissue culture plastic or in 3D collagen gels (5 mg/ml), induced with doxycycline for 64 hours and changes in let-7 and RNU48 (endogenous control) expression were analyzed by qRT-PCR from 3 separate experiments. *, p < 0.05 relative to control Panc1-tet-V cells. C. Mice were housed, fed and treated in accordance with the guidelines approved by the Northwestern University IACUC. Eight-week old athymic nu/nu animals (n=8) were subcutaneously injected with 5 × 106 Panc1-tet-V cells in the left flank and Panc1-tet-ΔC cells in the right flank of the same animal. Twenty-four hours later, doxycycline was added to the drinking water to induce MT1-MMP expression, and monitored twice a week for the development of skin tumors. The mice were euthanized, following which the tumors were dissected and placed in RNAlater (Qiagen, Valencia, CA). The RNAlater-preserved tumors were processed and analyzed for human MT1-MMP and human GAPDH mRNA or human let-7 and human sno135 microRNA expression by quantitative real-time PCR. For each animal the relative expression of let-7a, let-7d and let-7g was calculated in Panc1-tet-ΔC tumors and normalized to the levels present in the Panc1-tet-V tumors, with the let-7 expression in the control animals arbitrarily set at 1.0. Relative let-7 values < 1.0 indicate repression by MT1-MMP. D. Pancreatic tissue was obtained from patients with pancreatic adenocarcinoma and de-identified on an IRB-approved protocol. The cancerous and adjacent normal tissue samples were dissected and processed for RNA extraction using Trizol. The RNA quality was checked using Bioanalyzer-309 and samples with RNA Integrity Number (RIN) greater than 6 were used for RNA and microRNA studies. The let-7 levels relative to RNU48 and MT1-MMP expression relative to GAPDH in PDAC tumors (n=11) were normalized to the levels present in the matched adjacent normal pancreas, which was arbitrarily set at 1.0. Spearman’s correlation (GraphPad Instat) was used to determine significance between MT1-MMP and let-7 levels in these tumors.