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. Author manuscript; available in PMC: 2012 Feb 24.
Published in final edited form as: Oncogene. 2010 Nov 8;30(8):1002–1008. doi: 10.1038/onc.2010.485

Figure 1. Collagen repression of let-7 microRNA involves MT1-MMP.

Figure 1

Panc1 cells were grown on tissue culture plastic or in 3D type I collagen gels (5 mg/ml) in serum containing medium for 24 hours. A. MicroRNA were isolated using mirVana kit (Applied Biosystems, Foster City, CA) and analyzed for expression of the mature let-7a, let-7d and let-7g by qRT-PCR, with RNU48 as the endogenous control, using gene specific Taqman probes (Applied Biosystems, Foster City, CA). The data were quantified by the comparative CT method for relative gene expression from 3 experiments. *, p < 0.05 relative to cells grown on plastic. B. Similarly, MT1-MMP and GAPDH (endogenous control) mRNA were analyzed by qRT-PCR. *, p < 0.05 relative to cells grown on plastic. Data are an average from 3 separate experiments. C. Panc1 cells were grown on plastic or in 3D type I collagen gels (5 mg/ml) and treated with either DMSO or GM6001 (10 μM; EMD, Gibbstown, NJ) for 24 hours. MicroRNA were isolated and analyzed for let-7 and RNU48 (endogenous control) expression by qRT-PCR. *, p <0.05 relative to DMSO-treated cells grown in collagen. Data are an average of 3 separate experiments. D. Panc1 cells were transiently transfected with siRNA against MT1-MMP or control siRNA (50 nmoles) using Nucleofector Kit R (Lonza, Walkersville, MD). Cells were allowed to recover overnight and then plated on plastic or in collagen gels for 24 hours. MT1-MMP knockdown was analyzed by immunoblotting for cells grown on plastic (upper panel), while microRNA were analyzed for let-7 and RNU48 (endogenous control) expression by qRT-PCR. * p< 0.05; **, p < 0.01 relative to siControl transfected cells grown in collagen. Data are an average of 3 separate experiments.