Repression of Tumors in miR-143 Transgenic Mice Figure 1. Establishment of CAG/miR-143 transgenic mice and Northern blot analysis. A) Schematic depiction of the DNA construct and its possible transcript. ,2.6 kb SalI and HindIII fragment of CAG/miR-143 was injected to mice eggs. The shadow boxes and a bent arrow represent the 59untranslated region and a possible transcriptional start site, respectively. Approximate positions of primers for genotyping (arrowheads) are shown. B) Polyacrylamide gel Northern blot analysis of various tissues of the transgenic mouse. Twenty mg of total RNA of various tissues from a transgenic mouse (Line C) and its normal littermate was applied in each lane. The membrane was hybridized with the probes for miR-143(1st panel), miR-145 (2nd panel) and 5S rRNA (3rd panel). The 4th panel represents quantitative analysis of miR-143 expression in the 1st panel. SI: small intestine. doi:10.1371/journal.pone.0042137.g001 [21]. Since ERK5 is activated by MEK5, we tried to examine the effect of ERK5 activation on c-Myc expression by introducing dominant-negative MEK5A or constitute active MEK5D into human colon cancer cells DLD-1 [22]. As shown in Fig. 5A, the constitutive active MEK5D enhanced the c-Myc expression, whereas the dominant-negative MEK5A rather decreased it. Comparable results were also obtained in human embryonic kidney cells HEK293 (Fig. S3A). Hence, ERK5 activation might stabilize c-Myc protein. Intriguingly, Lee et al. recently demon- strated that activation of another member of the MAP kinase family, ERK stabilized c-Myc protein by phosphorylation in ApcMin/+ mice, suggesting the potential consequence of c-Myc stabilization by the MAP kinases in their tumorigenesis [23]. Next, we examined the effect of miR-143, miR-145 or siRNA for ERK5 on c-Myc expression in DLD-1 cells. Consistent with the results of MEK5 transfection, siRNA for ERK5 clearly repressed c-Myc expression (Fig. 5B, 1st and 2nd panels), suggesting that ERK5 could be one of the molecules through which miR-143 regulates c-Myc. c-Myc expression was not significantly suppressed by either miR-143 or miR-145 mimic when individually transfected. However, when expressed in combination, the effect was much pronounced. (Fig. 5B, 1st panel). It should be noted that miR-143 mimic significantly downregulated the expression of ERK5 although it was weaker than the effect of siRNA for ERK5 (Fig. 5B 2nd panel). Since Sachdeva et al. recently revealed that miR-145 when activated by p53 directly suppressed c-Myc expression, it is likely that miR-143 and miR-145 complement each other to downregulate c-Myc expression in our experimental systems [24]. This synergistic effect of miR-143 and miR-145 on c- Myc downregulation was also observed in another human colon cancer cell line, Lovo (Fig. S3B, 1st panel). p68/p72/b-catenin Signaling was Suppressed in the Transgenic Small Intestine Tumors DEAD-box RNA helicase subunits p68/p72, which are components of Microprocessor, promote the processing of pri- miR-143 and pri-miR-145 [13,14]. Thus, we investigated their expression in transgenic tumors. Unexpectedly, both the expres- sion p68 and p78 was drastically inhibited in the small intestine tumors of Tg/APC (Fig.4B 5th and 6th panels). This finding was exciting, since a recent study elegantly proved that p68/p72 were critically involved in b-catenin signaling in colon cancers [15]. Interestingly, c-Myc,cyclin D1 and c-jun are also the prominent targets of b -catenin/T-cell factor. PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42137