Many studies have demonstrated previously that the TGF-β pathway plays a critical role in breast cancer metastasis (18
). Several TGF-β/Smad-regulated genes have been shown to mediate TGF-β signaling in the control of cellular processes associated with breast cancer metastasis (18
). In this study, we report an miRNA expression signature of TGF-β-induced EMT in NMuMG epithelial cells. Twenty-eight miRNAs were found to be significantly deregulated by TGF-β in parental but not Smad4 knockdown NMuMG cells. Further, we showed that miR-155, the most significantly upregulated miRNA, plays an important role in TGF-β-induced EMT and cell migration and invasion. In addition, RhoA was negatively regulated by miR-155 and the restoration of RhoA in miR-155-overexpressing cells decreased TGF-β/miR-155-induced tight junction dissolution. Moreover, high expression levels of miR-155 correlate with invasive breast carcinomas. These findings are important for several reasons. First, they provide an miRNA expression signature of the TGF-β/Smad pathway in mammary gland epithelial cells. Second, the findings of this study establish a direct link between TGF-β/Smad4 and miR-155 during EMT. Finally, this is the first study to describe RhoA as a direct target of miR-155.
miR-155, a product of the BIC gene, is overexpressed in a number of human malignancies, which include B-cell lymphoma and carcinomas of the breast, colon, lung, and ovary (8
). Eμ-mmu-miR-155 transgenic mice develop B-cell malignancies (4
), whereas miR-155 knockout mice exhibit impaired immune function (40
). The transcription factor Pu.1 has been validated previously as a direct target of the miR-155-mediated immunoresponse (47
). Moreover, miR-155 represses tumor protein 53-induced nuclear protein 1 (TP53NP1), leading to pancreatic tumor development (10
). In addition, the NF-κB and AP-1 transcription factors have been shown previously to regulate miR-155 expression (20
). However, the functions of miR-155 in cell migration and invasion have not been investigated. Thus, our study provides the first evidence that miR-155 is upregulated by the TGF-β/Smad4 pathway and mediates TGF-β-induced EMT and cell invasion.
Previous computational and experimental studies have focused on the quality of sequence matching between miRNA and the target (6
). miRNAs negatively regulate their target mRNAs through base-pairing interactions, which lead to either mRNA degradation or translational inhibition, depending on the degree of matching between the seed sequence (positions 2 to 7 on the 5′ side) of miRNA and the 3′ UTR of mRNA; e.g., miRNA induces mRNA degradation when the seed sequence perfectly matches the target 3′ UTR or inhibits translation when the sequences are partially identical (6
). In addition, recent reports indicate that mRNA secondary structures may contribute to target recognition due to the fact that there is an energy cost associated with the un-base pairing of the messenger required to make the target site accessible for miRNA binding (27
). Kertesz et al. showed that site accessibility is as important as base pairing within the seeding region. Effective miRNA function requires nucleic acids flanking the target site, as well as the target itself, to be unpaired in a thermodynamically stable fashion (19
). Through the RNA22 algorithm, we found that the seed sequence of miR-155 has the potential to bind in multiple regions within the 3′ UTR of the RhoA gene. Of these sites, we selected three sites that fit the above-mentioned criteria and are highly conserved among species. Further, RhoA gene 3′ UTR reporter assays showed that miR-155 at all three sites significantly diminished luciferase activity. To mimic endogenous conditions more closely, we cloned the full-length RhoA gene 3′ UTR and performed experiments using TGF-β instead of the ectopic expression of miR-155. As expected, the full-length 3′ UTR responded to TGF-β, which was inhibited by the knockdown of miR-155 in the parental NMuMG line but not in the Smad4 knockdown line.
RhoA is the prototypical member of the Rho GTPase family, which regulates many cellular processes, including cellular adhesion, motility, and polarity, and is an important modulator of cell junction formation and stability (34
). Previous studies showed that TGF-β induces the disruption of tight junctions, cell polarity, and EMT through the ubiquitination and degradation of RhoA by Smurf1 E3 ligase that is activated by Par6 (34
). Our study demonstrated that TGF-β downregulated RhoA protein expression through the upregulation of miR-155 and thus provided an additional molecular mechanism of TGF-β regulation of RhoA (Fig. ). Regulation by miRNAs provides a means for cells to prevent protein translation, a mechanism to quickly prevent the accumulation of proteins by translational inhibition; our findings go hand in hand with earlier findings that TGF-β ubiquitinates RhoA for degradation. In this scenario, the induction of miR-155 halts the translation of RhoA while ubiquitination degrades translated RhoA proteins. Based on computational program predictions, each miRNA may negatively regulate hundreds of protein-encoding mRNAs (6
). Two recent studies using gene expression microarray analyses showed that miR-155 and its viral orthologue Kaposi's sarcoma-associated herpesvirus miR-K12-11 negatively regulate more than 180 mRNAs, some of which encode proteins involved in cell migration and invasion, including GSK3, PCSK5, and Rho GTPase-activating protein 21 (11
). Thus, RhoA is a major but not the only target that mediates miR-155 function in the control of cell polarity, EMT, and cell invasion contributing to cancer metastasis.
In addition, a previous study described a profile of miRNA expression in human keratinocytes treated with TGF-β. Four miRNAs were upregulated and another four were downregulated by TGF-β (55
). Of the eight deregulated miRNAs, only one miRNA (e.g., miR-21) showed a change consistent with our results. This discrepancy may be due to the use of different cell types and the duration of TGF-β treatment. Recent reports have indicated the importance of downregulation for miR-200 family miRNAs during TGF-β-induced EMT (2
). In congruence to previous findings, our array also showed the downregulation of miR-200c and miR-205 during a mesenchymal transition (Fig. ). However, the remaining members of the miR-200 family were not detected in our array. This result may be due to the use of different cell lines for the miRNA array analyses in our study and those described in previous reports (2
). In addition, we showed frequent upregulation of miR-155 in primary invasive breast cancer tissues. Consistent with this finding, a previous study reported that miR-155 is elevated in a metastatic breast cancer cell line, MDA-MB-231, but not in a nonmetastatic line, MCF-7 (28
). We also observed that the knockdown of miR-155 inhibited the TGF-β downregulation of E-cadherin but that the ectopic expression of miR-155 enhanced the TGF-β effects on E-cadherin expression (Fig. ). Sequence analysis showed no match between the seed sequence of miR-155 and the 3′ UTRs of the E-cadherin, ZEB1, and SIP1 genes (12
). Further investigation is required to determine the mechanism of miR-155 downregulation of E-cadherin, although it is likely that the effects are indirect but serve as a useful EMT indicator.
In summary, we demonstrated the miRNA expression signature of TGF-β/Smad-induced EMT in mammary epithelial cells. All 28 deregulated miRNAs contained at least one Smad4-binding site within their putative promoters. miR-155 mediated TGF-β/Smad pathway-induced EMT and cell migration and invasion through the targeting of RhoA. Further, the expression of miR-155 was associated with the invasive phenotype of breast cancer. Thus, miR-155 may be a potential metastatic/prognostic marker and therapeutic target for breast cancer metastasis intervention.