Impaired microRNA processing causes corpus luteum insufficiency and infertility in mice
J. Clin. Invest. Motoyuki Otsuka, et al. 118:1944 doi:10.1172/JCI33680 [Go to this article.]

Figure 4
miR17-5p and let7b can target 3′-UTR of mTIMP1. (A) miRNA target positions in the 3′-UTR of mTIMP1 were identified by computational prediction methods, MicroInspector, and miRanda. The same candidate miRNAs, miR17-5p and let7b, were predicted by the different programs; however, the predicted miRNA-targeting sequences in the 3′-UTR of TIMP1 are not exactly the same. (B) Primer extension analyses showed the expression of the indicated miRNAs in Dicer^+/+ and Dicer^d/d mice ovaries. NC, negative control. (C) miR17-5p and let7b can target 3′-UTR of TIMP1. The 293T cells were transiently transfected with a reporter plasmid (pLuc-TIMP3′-UTR) with or without indicated miRNA expression plasmids, and 36 hours after transfection, a reporter assay was performed. The relative luciferase values were calculated by dividing firefly luciferase values with internal control renilla luciferase values. The value from the negative control was set at 1. Data are shown as mean ± SD from 3 independent experiments. *P = 0.032, **P = 0.00082, ***P = 0.0035. (D) miR17-5p and let7b downregulate TIMP1 expression and activity. The 293T cells were transiently transfected with pcDNA-TIMP1, with or without the indicated miRNA expression plasmids, and pRL-TK. TIMP1 expression was examined by western blotting 36 hours after transfection (top). Cell extracts were normalized based on the luciferase value of cotransfected pRL-TK to avoid the variances of transfection efficiency. Bottom: Result of reverse zymography using the MMP2-containing gels and the concentrated culture supernatant collected from the transfected cells to quantitate the activities of TIMP1 against MMP2 activities. A representative result from 4 independent experiments is shown. Numbers below the images refer to the fold changes of the intensities.