05). However, as TIMP3 mRNA expression was very low in each of the four cell lines, a significant correlation LDN-193189 in vitro between miR-21 and TIMP3 mRNA was not detected (data not shown). Figure 3 TIMP3 protein expression correlates with microRNA-21 content in breast cancer cell lines in vitro. A, Western blot analyses of TIMP3 protein, performed

as described in Methods. B, Correlation between miR-21expression and TIMP3 protein levels (Pearson correlation = -0.905; P < 0.05). The TIMP3 3'-UTR is a target for miR-21 To determine whether suppression of miR-21 impacts TIMP3 transcription, we quantified TIMP3 mRNA in MDA-MB-231 and MDA-MB-435 cells (each expressing high levels of endogenous miR-21) following knockdown of miR-21 expression. Down-regulation of endogenous miR-21 (Fig. 4A) led to a 1.3 and 1.4 fold increase in TIMP3 mRNA in MDA-MB-231 and MDA-MB-435 cells, respectively Ilomastat molecular weight (Fig. 4B). Similar increases in TIMP3 protein expression following miR-21 knockdown were observed (Fig. 4C, 4D). These data suggest that TIMP3 is regulated

by miR-21 in breast cancer cells. In order to determine whether the 3′untranslated region of TIMP3 PD173074 clinical trial mRNA is a direct functional target of miR-21, we cloned a 250 bp TIMP3 3′-UTR segment, which includes a potential target site for miR-21 (Fig. 4E), downstream of the pGL3 luciferase reporter gene to generate the pGL3-timp3 vector. This vector was co-transfected into MDA-MB-435 or MDA-MB-231 cell lines together with anti-miR-21 oligonucleotides or miRNA negative control. A renilla luciferase vector (pRL-TK) was used to normalize differences in transfection efficiency. Luciferase activity in MDA-MB-435 cells co-transfected with pGL3-timp3 vector and anti-miR-21 oligonucleotides significantly increased by 38% when compared with negative control (P < 0.05), whereas luciferase activity in MDA-MB-231 cells increased by only 20% (Fig. 4F). These data demonstrate Sorafenib order that miR-21 regulates TIMP3 expression at the transcriptional level. 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.