Repression of Tumors in miR-143 Transgenic Mice Figure 4. Western blot analysis of the gut tumors. A) Whole cell extracts of the small intestine and colon tumors from Tg/APC and their non- transgenic littermates (W/APC) were examined by anti-ERK5 antibody. B, C) Whole cell extracts of the small intestine tumors (B) and the colon tumors (C) from Tg/APC and their non-transgenic littermates (W/APC) were examined by the indicated antibodies. Lower and upper arrows indicate p72 and its alternatively spliced form p82, respectively. doi:10.1371/journal.pone.0042137.g004 demonstrated that miR-145 mimic decreased reporter activity of vector with the wild 39UTR of p72 by 73% of that of the mutated 39UTR. These results implied that the downregulation of p72 observed in the transgenic small intestine tumors could be at least in part a direct effect of miR-145. As far as we examined, none of miR-143 and miR-145 mimics significantly inhibited p68 expression in DLD-1 cells and Lovo cells (Fig. 5B, 4th panel, Fig. S3B, 3rd panel). Heinlein et al. [25] and Ro¨ssler et al. [26] revealed that the 59region of p68 of mouse and human had a canonical E-box, respectively. The latter group furthermore showed that the deletion of the E-box reduced the reporter activity by half. Thus, c-Myc may be one of the candidates that positively regulate p68 expression. To address this hypothesis, we introduced three distinct c-Myc siRNAs into DLD-1 cells and analyzed the expression level of p68. As shown in Fig. 5C, these three siRNAs, all of which strongly suppressed the expression of c-Myc, decreased that of p68. These data implied that failure of suppression of p68 expression by mimics of PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42137