Repression of Tumors in miR-143 Transgenic Mice Figure 3. RNA analysis of gut tumors in the transgenic mice. A) Polyacrylamide Northern blot analysis of expression of miR-143 and miR-145 of gut tumors. Ten mg of total RNA of small intestines and colons of Tg/APC and their non-transgenic littermates (W/APC) was applied in each lane. The membrane was hybridized with the probes for miR-143(upper panel), miR-145 (middle panel) and 5S rRNA (lower panel). Individual identification number is presented. B) Quantitative Real-Time PCR (qRT-PCR) analysis of the mouse endogenous pri-miR-143 and pri-miR-145. The expression of mouse pri-miR-143 and pri-miR-145 of the small intestine tumors from Tg/APC (gray bars, n = 5) and their non-transgenic littermates (W/APC) (open bars, n = 4) was examined. Each set of primers cover the region that contains pre-miR-143 or pre-miR-145. The data are representative of three independent experiments and are presented as the mean 6 SEM. C) qRT-PCR analysis of the mouse endogenous pri-miR-143 and pri-miR-145 of the colon tumors. The expression of mouse pri-miR-143 and pri-miR-145 of tumors from Tg/APC (gray bars, n = 3) and their non-transgenic littermates (W/ APC) (open bars, n = 3) was examined in the same way as B). doi:10.1371/journal.pone.0042137.g003 miR-145. miR-145 also suppressed the expression of p78 in Lovo cells (Fig.S3B, 2nd panel). Analysis by TargetScan indicated that there existed one possible binding site of miR-145 in the 39untranslated region (39UTR) of p72 of many animal species. Thus, miR-145 might directly bind the mRNA of p72 and restrain the expression of p72. To test this idea, we constructed a fusion gene of luciferase reporter gene and the p72 39UTR harboring one miR-145 target sequence, and subjected it to reporter assays. A construct with mutations in the seed sequence was made for a negative control (Fig. 5D). Fig. 5E PLoS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42137