We also analyzed the signaling in the non-tumorous segments of transgenic small intestines. As shown in Figure S4A, the expression level of miR-145, as well as normal organs (Fig. 1B), was almost comparable between the transgenic mice and their littermates. Although the expression of ERK5 in non-tumorous segments of Tg/APC small intestines was significantly decreased, retardation of cyclin D1, c-jun and c-Myc expression was subtle or much weaker compared to that in tumors (Fig. S4B, 1st-4th panels). The expression of p68 in non-tumorous segments of Tg/APC was inhibited but more slightly than in tumors though its overall expression level seemed to roughly correspond to that of c-Myc (Fig. S4B, 5th panel), and the suppression of p72 was hardly detected (Fig. S4B, 6th panel). These data suggested that forced expression of miR-143 should be sufficient for ERK5 repression, but that miR-145 induction might be a crucial event for full inhibition of cyclin D1, c-jun and c-Myc through retardation of 68/p72/b-catenin signaling. Consistent with the ERK5 expression level, c-Myc and p68/p72 were expressed in colon tumors of Tg/APC at similar levels as W/ APC, although the expression of p72 in some samples of Tg/APC was weak (Fig.4C). Possible Involvement of c-Myc in the Elevation of miR- 145 Expression To examine whether miR-143 induces the miR-145 expression in human colon tumor cells, we introduced miR-143 mimic into DLD-1 cells and Lovo cells, and analyzed the miR-145 expression by qRT-PCR. There was, however, no obvious enhancement of miR-145 (data not shown). Indeed, although miR-143 was strongly expressed in one colon tumor of transgenic mice, the induction of miR-145 was poor (Fig.3A #13). Hence, additional events other than miR-143 expression seemed necessary for substantial induction of miR-145 in colon cancer cells. c-Myc is an essential transcription factor in development of tumors and its expression was decreased in the tumors of the intestine but not the colon in Tg/APC. Intriguingly, c-Myc has already been proven to form the feedback loop with miR-17-92 cluster in cancer cells [27].Thus we introduced siRNA for c-Myc into DLD-1 and Lovo cells. Figure 5F showed that c-Myc siRNA enhanced the expression of miR-145 to some extent in both cells. Moreover, miR143 expression also increased by c-Myc siRNA (Fig. 5G). To examine whether the regulation of miR-145 by c-Myc occurred at the transcriptional level, we performed qRT-PCR analysis of pri-miR-145. Consistent with the transgenic intestine tumors, the expression of pri-miR-145 of c-Myc siRNA-intro- duced cells was increased (Fig 5H). These data implicated that suppression of c-Myc might be at least partly involved in the elevation of miR-145 and probably of miR-143 in the transgenic intestine tumors. Discussion Here, we present that forced expression of miR-143, which is in vivo processed from pri-miRNA, induces miR-145 expression and represses the small intestine tumor formation in ApcMin/+ mice. ERK5, also known as BMK1, is one of the MAP kinases and was shown to be involved in various types of cancers including prostate and breast cancers [28]. While its role in gut cancer development was not clear, the phosphorylation of ERK5 was increased by fetal bovine serum stimulation in Caco-2 cells [29]. Recently, ERK5 was found to be a target of miR-143 in adipocytes [19]. We demonstrated the expressions of ERK5 and its downstream effectors, c-Myc, c-jun and cyclin D1, were also suppressed in the Repression of Tumors in miR-143 Transgenic Mice transgenic small intestine tumors. In particular, c-Myc expression seemed to be tightly linked to the ERK5 expression level. Of interest, c-Myc has been proven to be a critical mediator of b-catenin signaling in tumorigenesis of intestines after APC loss [23,30,31]. The data of our transfection experiments, together with these previous studies, suggest that miR-143 probably inhibits tumor development by downregulating c-Myc expression in cooperation with miR-145 in our transgenic mice. Downregulation of p68/p72 could also be one of the key factors that retard tumor development in the small intestine of Tg/APC. Our results suggested the downregulation of p72 by miR-145 through binding to its 39UTR might be at least one of the molecular basis for the decrease of its expression in the transgenic small intestine tumors. In addition, we showed that the expression of p68 was downregulated by the introduction of c-Myc siRNAs. Thus, it is possible that p68 and c-Myc might form a positive feedback loop, which suggests that miR-143 and miR-145 might inhibit p68 in part through the repression of c-Myc. Further analysis by TargetScan also revealed the existence of possible target sites of miR-206 and miR-34a/miR-206/miR-26a in the 39UTRof p68 and p72, respectively, in a wide range of animal species. This was intriguing, since miR-206 and miR-26a are a set of miRNAs whose processing was proven to depend on the p53/p68/p72 complex [14]. In addition, miR-34a is a well- known p53-induced gene [32], and p68 and p72 have been shown to be strong and weak co-activators of p53, respectively [33]. Crucially, all of these were miRNAs that were found to be tumor suppressors [34,35]. Indeed, our reporter assays showed that miR-34a (Fig. S5 B and F), miR- 206 (Fig. S5 C and G), and miR-26a (Fig. S5 D and H) mimics suppressed the reporter activity of the constructs contain- ing each target on the p72 39UTR, whereas miR-206 mimic also decreased that of the construct containing a target on the p68 39UTR (Fig. S5 A and E). Although further studies are required, the p53/p68/p72 complex and a set of tumor suppressor miRNAs linked with p53 seem to constitute a novel regulatory feedback circuit. Whereas miR-145 was induced in the small intestine tumors in Tg/APC, the molecular mechanisms remain to be obscure. Our data using DLD-1 cells and Lovo cells, however, imply that the suppression of c-Myc may be at least in part involved in its induction. Hence, given qRT-PCR analysis of pri-miR-143/145 and Western blot analysis of transgenic tumors, forced-expression of miR-143 might trigger c-Myc/pri-miR-145 signal more easily in the small intestine tumors than in the colon tumors. Interestingly, this signaling axis appeared not to exert its function efficiently in non-tumorous segments of small intestines. Other investigators recently reported that a positive feedback circuit between miR-143 and miR-145 could work through suppressing KRAS-RREB1 signaling in pancreatic cancer cells, although they did not mention the cross-regulation at the mature miRNA levels [36]. Of note, they showed, however, this circuit was observed only in KRAS mutant cells but not in KRAS wild cells. Since KRAS mutation is not a usual event in tumors of ApcMin/+ mice [9,10], the induction of miR-145 by miR-143 in our transgenic mice would be dependent on a molecular mechanism distinct from KRAS-RREB1 signaling. Nonetheless, our study with their report indicates that regulatory circuits between miR- 143 and miR-145 might exert anti-tumor effect in a variety of neoplasms in living animals. The incidence of transgenic colon tumor development, which exhibited poor miR-143 expression, unexpectedly increased compared to non-transgenic littermates. Of interest, this result PLoS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e42137