Figure 4

miR-21 regulates TIMP3 expression at the mRNA and protein level by targeting the 3'untranslated region of TIMP3 mRNA. A, miR-21 expression was analyzed by TaqMan PCR in MDA-231 and MDA-435 cells following transfection with anti-miR-21 or control oligonucleotides, as in Fig. 2B. B, Relative TIMP3 mRNA expression was analyzed in MDA-231 and MDA-435 cells as described in Methods, following miR-21 silencing as performed in A. C, Western blot analysis of TIMP3 protein expression in MDA-231 and MDA-435 cells following miR-21 silencing as performed in A. D, Quantification of relative TIMP3 protein expression in MDA-231 and MDA-435 cells following miR-21 silencing, as performed in A. E, Generation of cDNA encoding the 3'UTR region of TIMP3 containing a miR-21 binding site. cDNA was subsequently cloned into a Luciferase reporter plasmid. F, Determination of the impact of miR-21 silencing on pGL3-TIMP3 luciferase expression in MDA-231 and MDA-435 cells. Data represents two independent experiments performed in triplicate.*, P < 0.05.