RNaseIII endonuclease protein family, Drosha and Dicer [11]. Another RNA binding protein, DGCR8, has been found to interact with Drosha and to be a requisite for the processing of pri- miRNAs. Together with other RNA binding molecules such as DEAD-box RNA helicase subunits p68/p72 (also known as DDX5 and DDX17, respectively), Drosha and DGCR8 form a huge complex, Microprocessor [12]. Indeed, the p68/p72 knockout mice study demonstrated that p68/p72 were required for the processing of pri-miRNAs of a subset of miRNAs, such as miR-145 and miR-16 [13]. Moreover, Suzuki et al. reported that the processing of pri-miR- 143 and pri-miR-145 required the interaction of the tumor suppressor p53 and the Drosha complex through the association with p68/p72 in colon cancer cells, suggesting that the full expression of miR-143 and miR-145 might be involved in tumor suppressing signaling driven by p53 [14]. On the other hand, accumulating evidence has shown a possible implication of p68/p72 for b-catenin signaling in gut tumor development. The interaction of p68/p72 and b-catenin could play a pivotal role in Wnt/b-catenin signaling, to activate the downstream effectors such as c-Myc, cyclin D1 and c-jun [15]. PDGF-induced tyrosine-phosphorylated p68 associated with b- catenin and promoted the epithelial-mesenchymal transition of human colon cancer cells, suggesting the potential implication of p68 in colon cancer metastasis [16]. Here, we establish transgenic mice in which miR-143 is ubiquitously expressed in a variety of organs. When crossbred with these mice, the development of small intestine tumors of ApcMin/+ mice is retarded. Interestingly, endogenous miR-145 is also increased in these tumors. Molecular examination shows that protein expression of extracellular signal regulated kinase (ERK5), p68/p72 and c-Myc is strongly suppressed. We also present that the expression of c-Myc and p72 is downregulated by miR-143/ miR-145 and miR-145, respectively, in a human colon cancer cell lines, DLD-1 and Lovo cells. The reporter assay shows that p72 could be a direct target of miR-145. As far as we are aware, this is the first report that miR-143 suppresses tumors spontaneously developing in living organisms. This study may also provide a unique model where tumor suppressive miRNAs and the key regulators for their biogenesis, p68/p72, form a regulatory circuit. Results Forced Expression of miR-143 Induced miR-145 Expression in the Small Intestine Tumors of ApcMin/+ Mice and Suppressed the Tumor Development To express miR-143 ubiquitously in whole body, we made a construct which carried ,300 bp human pri-miR-143 fragment under the CAG regulatory unit, composed of CMV enhancer and chicken b-actin promoter, and injected it into the fertilized mice eggs [17] (Fig.1A). We obtained four founder mice, and three of them transmitted the transgene to offspring. Since only one strain (Line C) strongly expressed miR-143, we used this strain for further analysis (Fig.1B and Fig. S1A). The transgenic mice of Line C have no abnormality in appearance. After backcrossing the mice to C57BL/6 mice four times, we crossbred the transgenic mice with ApcMin/+ mice and dissected 4 month old mice to examine the tumor development (Fig. S1B). Interestingly, the small intestine tumor incidence was significantly suppressed in ApcMin/+ mice carrying the transgene (hereafter referred to as Tg/APC) (Fig. 2A, 2B, 2C, 2D, 2E). On the other hand, the colon tumors developed in Tg/APC at higher frequency than non-transgenic littermates (hereafter referred to as Repression of Tumors in miR-143 Transgenic Mice W/APC) (Fig. 2F and 2G). As far as we examined, all the tumors of Tg/APC were adenomas and histologically showed no apparent difference from those of W/APC (Fig. S2). As shown in Fig.3A, miR-143 was highly expressed in the small intestines tumors of Tg/APC whereas the colon tumors generally expressed lower. Thus, the sufficient expression of miR-143 appears to restrain tumor development in living animals. Unexpectedly, the expression of miR-145 of transgenic small intestine tumors also increased in proportion to that of miR-143 (Fig. 3A). In contrast, we detected only little enhancement of miR- 145 in Tg/APC colon tumors even though one of the tumors examined strongly expressed miR-143 (see Fig.3A #13). Thus, the induction of endogenous miR-145 appears to work preferentially in the small intestine polyps. miRNAs are initially transcribed as several kilo bp pri-miRNAs, which are finally cleaved into mature miRNAs through the intermediates, ,65 bp stem-loop precursor miRNAs (pre-miR- NAs). miR-143 and miR-145 were transcribed as a bicistronic unit, a common pri-miRNA, in DGCR8-null embryo bodies [18]. To examine whether the upregulation of miR-145 in Tg/APC tumors was due to the increase of pri-miRNA, we performed qRT-PCR analysis of the mouse endogenous pri-miR-143 and pri- miR-145 with two sets of primers covering each pre-miRNA region. Figure 3B indicated that both of pri-miR-143 and pri-miR- 145 expression was upregulated in the small intestine tumors of Tg/APC. Thus, the forced expression of miR-143 in the small intestine tumors may induce the transcription of a bicistronic pri- miR-143/miR-145 to promote the expression of miR-145, and possibly miR-143. Since no techniques, however, have been available to discriminate the human and the mouse mature miR- 143, we could not confirm an auto-regulatory loop of miR-143. On the other hand, neither of pri-miR-143 nor pri-miR-145 significantly increased in the transgenic colon tumors (Figure 3C). Hence, the failure of miR-145 induction in transgenic colon tumors seemed to occur at the transcriptional level. ERK5-c-Myc Signaling was Suppressed in the Transgenic Small Intestine Tumors Next, we tried to identify the direct target molecule of miR-143 which would contribute the retardation of small intestine tumors. So far, several candidates for miR-143 targets in cancer cells have been identified. Extracellular signal regulated kinase 5 (ERK5) has been the most intensively studied so far. Esau et al. [19] initially documented ERK5 as a target in differentiating adipocytes and other investigators confirmed this notion in some human and rodent cancer cells [5,20]. We examined the ERK5 expression in tumors by Western blotting. Corresponding to the miR-143 expression, ERK5 expression in the small intestine tumors of Tg/APC was downregulated compared to W/APC, whereas no significant difference was observed in the colon (Fig. 4A). Thus, a significant level of miR-143 expression might be necessary for ERK suppression, or colon cells might have low sensitivity of ERK5 to miR-143. Then, we examined the expression of cyclinD1 and c- jun that have been shown to be the downstream effectors of ERK5 signaling. As shown in Fig. 4B (1st and 2nd panels), both expression was downregulated in the transgenic small intestine tumors. c-Myc plays a central role in regulation of the proliferation of a variety of cells. Interestingly, the level of c-Myc was closely related to that of ERK5 (Fig. 4B 3rd and 4th panels), strongly implicating that c-Myc was one of the major downstream targets of miR-143 through ERK5. Although c-Myc was proven to be a substrate of ERK5, it was not clear whether the expression of c-Myc was regulated by ERK5 PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42137