Identification of EMT-associated miRNAs

To identify regulatory miRNAs involved in the gradual process of an EMT, we performed miRNA sequencing on a detailed time course of a TGFβ-induced EMT in normal murine mammary gland cells (NMuMG subclone E9; NMuMG/E9). Analysis of the kinetics of miRNA transcript regulation during an EMT in a time-resolved manner identified 32 differentially expressed miRNAs. Unsupervised hierarchical clustering illustrated that approximately half of the differentially expressed miRNAs showed a continuous increase in their expression during an EMT, whereas the other half exhibited decreased expression (Fig. 1a). In order to identify the miRNAs functionally impacting on a TGFβ-induced EMT, we performed a microscopy-based screen in which NMuMG/E9 cells were transfected with miRNA mimics and cultured in the absence or presence of TGFβ for 4 days (Fig. 1b). Subsequently, mesenchymal cell characteristics were monitored by high-content fluorescence microscopy analysis and quantified, including the deposition of the extracellular matrix protein fibronectin and the formation of focal adhesions and of actin stress fibres23. Nine out of 32 differentially expressed miRNAs were able to block or at least delay the EMT process: miR-125b-5p, miR-181b-2-3p, miR-1247-3p, miR-200a-3p, miR-200b-3p, miR-429-3p, miR-1199-5p, miR-145a-3p and miR-504-5p. Conversely, the forced expression of miR-145a-5p and miR-6944-3p promoted an EMT in epithelial NMuMG/E9 cells. Changes in cell morphology, the localization of the epithelial adhesion junction protein E-cadherin as well as the mRNA levels of E-cadherin, N-cadherin, fibronectin and Zeb1 further confirmed the impact of the different miRNAs on the EMT process (Supplementary Fig. 1a–c).

Fig. 1 Identification of miRNAs critical for an EMT and cell migration. a Expression profiling of miRNAs during a TGFβ-induced EMT in NMuMG/E9 cells. NMuMG/E9 cells were treated with TGFβ for the time points indicated, and total RNA was isolated for miRNA sequencing. The heat map summarizes the hierarchical clustering of differentially expressed miRNAs (log2FC(±2); FDR <0.05) compared to epithelial, untreated cells during an EMT according to the indicated colour scale. b Identification of miRNAs controlling EMT. NMuMG/E9 cells were transfected with miRNA mimics for individual miRNAs and analysed for mesenchymal characteristics in the absence and presence of TGFβ for 4 days. Formation of focal adhesions (green), actin cytoskeleton reorganization to actin stress fibres (red), fibronectin deposition (yellow) and nuclei (blue) were visualized by high-content fluorescence screening microscopy. c Identification of miRNAs regulating mesenchymal tumour cell migration. Mesenchymal, migratory Py2T cells (>20 days TGFβ) were transfected with different miRNA mimics, plated in 96-well Boyden chamber migration inserts within a FCS gradient and in parallel on a 96-well input plate. Cell nuclei were imaged using a fluorescence screening microscope and quantified. Migrated cells were normalized to the total cell number on the input plate. d Scheme depicting the workflow and the number of (a) differentially expressed miRNAs during an EMT, (b) the number of identified miRNAs functionally contributing to a TGFβ-induced EMT and (c) the number and names of the miRNAs functionally contributing to mesenchymal tumour cell migration Full size image

Increased cell migration is a functional output of EMT, which allows tumour cell invasion and dissemination into the surrounding tissue1. In a second screening step, we identified those miRNAs that were able to affect mesenchymal tumour cell migration (Fig. 1c). Mesenchymal, highly migratory and tumorigenic Py2T cells that have been treated >20 days with TGFβ24 were transiently transfected with mimics of EMT-affecting miRNAs. Four out of the 11 miRNAs tested significantly reduced cell migration in a trans-well Boyden chamber assay: miR-200b-3p, miR-429-3p, miR-1199-5p, and to a lesser extent miR-125b-5p (Supplementary Fig. 2a, b). As a result, our experimental strategy identified miRNAs whose transcriptional regulation and function affected both a TGFβ-induced EMT and mesenchymal breast cancer cell migration (Fig. 1d). Four miRNAs significantly fulfilled these criteria: the miRNA-200 family members miR-200b-3p and miR-429-3p, and miR-125b-5p and miR-1199-5p. Since ectopic expression of miR-1199-5p, an EMT-regulatory miRNA, seemed to affect EMT with similar potency as the well-studied miR-200 family members15, 17, 18, we further investigated the functional role and the mechanisms of action of miR-1199-5p in the regulation of an EMT.

miRNA-1199-5p inhibits EMT and tumour cell invasion

The continuous downregulation of miR-1199-5p expression during a TGFβ-induced EMT was due to transcriptional repression as determined by a miR1199-promoter/luciferase-reporter assay during a TGFβ-induced EMT in NMuMG/E9 cells and Py2T murine breast cancer cells, and in mesenchymal Py2T cells and metastatic 4T1 murine breast cancer cells treated with TGFβ for >20 days (Fig. 2a, Supplementary Fig. 3a, b). Analysis of miR-1199 expression in different human breast cancer cell lines25 further confirmed a higher expression in epithelial than in mesenchymal breast cancer cells (Supplementary Fig. 3c).

Fig. 2 miRNA-1199-5p inhibits an EMT and tumour cell migration and invasion. a miR-1199-5p transcript and miR-1199 promoter activity levels decrease during a TGFβ-induced EMT. Green line: expression profile of miR-1199-5p during EMT in NMuMG/E9 cells as determined by RNA sequencing. Grey bars: miR-1199 promoter activity during an EMT. NMuMG/E9 cells were treated with TGFβ for the time points indicated and transfected with a Renilla luciferase reporter along with either a miR-1199 promoter Firefly luciferase reporter (pGL4 miR-1199 promoter) or a control reporter (pGL4; Supplementary Fig. 3b). Relative luminescence (Firefly/Renilla) was calculated and normalized to the control reporter (mean fold changes ± s.e.m.; n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05, **P < 0.01). b, c NMuMG/E9 cells were transiently transfected with the miRNA mimics indicated and cultured in presence of TGFβ for 4 days (4-day TGFβ). Bright-field images of NMuMG/E9 cells illustrate the differences in cell morphology. Immunofluorescence images of NMuMG/E9 cells visualize different epithelial and mesenchymal cell structures as indicated. Scale bars: 100 μm. d, e Immunoblot (d) and quantitative RT-PCR mRNA (e) expression analysis of NMuMG/E9 cells transiently transfected with miRNA mimics for the EMT markers indicated (mean fold changes ± s.e.m.; n = 6; significance determined by an unpaired, two-sided t test, Student’s t test; **P < 0.01, ***P < 0.001, ****P < 0.0001). f, g Mesenchymal (>20 days TGFβ) Py2T (f) and 4T1 (g) cells were transiently transfected with the miRNA mimics indicated and re-plated within a FCS gradient of a Boyden chamber migration or invasion insert. Nuclei of transmigrated and invaded cells were quantified after 18 h (mean fold changes ± s.e.m.; migration: n = 3; invasion Py2T: n = 3, 4T1: n = 4; significance determined by an unpaired, two-sided t test; *P < 0.05, **P < 0.01, ****P < 0.0001) Full size image

The forced expression of miR-1199-5p by the transfection of a construct encoding a 1199-5p miRNA mimic caused sustained epithelial cell morphology in NMuMG/E9 and in human untransformed mammary gland MCF10A cells induced to undergo an EMT by TGFβ treatment (Fig. 2b–e, Supplementary Fig. 3d–f). The cells maintained their epithelial morphology and the cell surface localization of the adherens junction protein E-cadherin and of the tight junction protein ZO-1 at the plasma membrane, while miR-Ctr-transfected cells lost these epithelial characteristics and progressed with an EMT (Fig. 2b, Supplementary Fig. 3d). The reorganization of cortical actin to stress fibres as well as the formation of focal adhesions were also prevented by miR-1199-5p mimics (Fig. 2c). Mesenchymal markers, such as fibronectin, vimentin or N-cadherin were repressed at the protein (Fig. 2d, Supplementary Fig. 3e) and mRNA (Fig. 2e, Supplementary Fig. 3f) expression levels in miR-1199-5p-transfected cells. Furthermore, miR-1199-5p induced a significant decrease in Zeb1 mRNA, however, transcript levels of other key EMT TFs, such as Zeb226 or Sox427 remained unchanged (Fig. 2e).

A gain in cell migration and invasion can be one of the consequences of a TGFβ-induced EMT and allows tumour cells to leave the primary tumour and intravasate into the blood circulation and to extravasate at a distant organ1, 5. Ectopic expression of miR-1199-5p displayed only minor effects on the early stages of an EMT in Py2T cells (Supplementary Fig. 3g–i), yet it induced a significant reduction in migration and invasion of mesenchymal (>20 days TGFβ) Py2T cells, as analysed by trans-well Boyden chamber assays (Fig. 2f). The inhibitory effects of miR-1199-5p on in vitro cell migration and invasion were also observed in 4T1 cells treated for >20 days with TGFβ (Fig. 2g). In summary, miR-1199-5p is sufficient to sustain an epithelial cell phenotype.

The direct target genes of miR-1199-5p during an EMT

To elucidate the genome-wide function of miR-1199-5p during an EMT, we performed RNA-sequencing analysis on NMuMG/E9 cells transiently transfected with either a miR-1199-5p mimic or a miR-Ctr mimic and cultured for 4 days in the presence of TGFβ. As expected, forced expression of miR-1199-5p induced a block in a TGFβ-induced EMT in these cells and led to an overall anti-correlative (r = −0.324) transcriptomic profile compared to genes regulated in their expression during an EMT (miR-Ctr 4d vs. miR-Ctr 0d) (Fig. 3a). We found 787 genes differentially expressed (log2 fold change of ±1 (log2FC(±1)); False Discovery Rate (FDR) <0.05) by the ectopic expression of miR-1199-5p during an EMT. Subsequent functional annotation analysis by DAVID (Database for Annotation, Visualization and Integrated Discovery)28, 29 for biological processes (GO (gene ontology)) and cellular pathways (Kyoto Encyclopedia of Genes and Genomes; KEGG) revealed their involvement predominantly in cell adhesion processes, extracellular matrix (ECM)-receptor interactions and focal adhesions (Fig. 3b).

Fig. 3 Targets of miR-1199-5p function during an EMT. a Overall gene expression regulation by miR-1199-5p during a TGFβ-induced EMT. RNA-sequencing analysis was performed on NMuMG/E9 cells transiently transfected with a miR-1199-5p or a negative control (miR-Ctr) mimic. Cells were cultured for 4 days in the presence (4 days; miR-Ctr- and miR-1199-5p-transfected cells) or absence of TGFβ (only miR-Ctr-transfected cells). The scatterplot depicts overall gene expression (log2FC) anti-correlation between miR-1199-5p 4 days vs. miR-Ctr 4 days over miR-Ctr 4 days vs. miR-Ctr 0 days. Differential expression analysis (red dashed line: log2FC(±1); False Discovery Rate (FDR) <0.05) identified 787 genes regulated by miR-1199-5p during an EMT. Green quadrant: increased gene expression; red quadrant: decreased gene expression. b Functional annotation clustering analysis of differentially expressed genes from a. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses by DAVID for the top five biological processes (top), pathways (bottom) and their associated P and Benjamini–Hochberg values. c Identification of miR-1199-5p direct targets in EMT. The Venn diagram depicts the number of genes differentially expressed upon miR-1199-5p expression during an EMT (blue), predicted direct targets of miR-1199-5p by miRWalk2.0 (red) and the number of overlapping genes. Sixty-six out of 90 genes demonstrate a downregulation in their expression upon forced expressed of miR-1199-5p in EMT, including the EMT TF Zeb1 Full size image

In order to delineate the mechanism by which miR-1199-5p maintains an epithelial cell morphology, we set out to determine its direct mRNA targets during an EMT. Computational analysis by miRWalk30 revealed 1789 potential target mRNAs of miR-1199-5p, which display a seed sequence in their 3′ UTR, 5′ UTR or coding sequence (CDS). Overlaying these mRNA targets with genes regulated by miR-1199-5p during an EMT (Fig. 3a; 787 genes) uncovered 90 target genes of which 66 displayed a significant reduction in their transcript levels upon the forced expression of miR-1199-5p (Fig. 3c, Supplementary Table 1). Among these genes, we identified the key EMT TF Zeb131 with a potential conserved 8-mer seed sequence for miR-1199-5p in its 3′ UTR (Fig. 4a).

Fig. 4 miR-1199-5p directly controls Zeb1 expression. a Species-conserved miR-1199-5p-binding site in the 3′ UTR of Zeb1. The scheme represents: top: sequence alignments of predicted binding sites of miR-1199-5p (red; mouse: position 239–246) in Zeb1 3′ UTRs of different species. Middle: alignment of the 8mer seed match in mouse miR-1199-5p. Bottom: mutated miR-1199-5p seed sequence in the 3′ UTR of Zeb1 mRNA utilized for the control reporter construct (Zeb1 3′ UTR mut) in d. Exchanged nucleotides are underlined. b, c Forced expression of miR-1199-5p reduces Zeb1 nuclear localization and protein levels during an EMT. NMuMG/E9 cells were transiently transfected with miRNA mimics as indicated and cultured in the absence (0 day) or presence of TGFβ (4 days). Immunofluorescence (b) and immunoblotting (c) analyses illustrate the differences in Zeb1 protein levels. Scale bar: 100 μm. d Post-transcriptional regulation of Zeb1 by miR-1199-5p. NMuMG/E9 cells were transfected with the miRNA mimics indicated, with a Renilla luciferase reporter construct and with either a Zeb1 3′ UTR wild-type (wt) or a Zeb1 3′ UTR mutant (mut) Firefly luciferase reporter construct. Relative luminescence (Firefly/Renilla) was calculated and normalized to miR-Ctr-transfected cells (mean fold changes±s.e.m.; n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05) Full size image

miR-1199-5p directly targets Zeb1 mRNA

The zinc-finger E-box-binding homeobox TF Zeb1 has a well-established role as transcriptional repressor of epithelial genes and, hence, as an activator of an EMT3, 31. Zeb1 function is associated with increased stemness, cell survival and metastasis32, 33, and high levels of Zeb1 have been linked to aggressive breast cancer subtypes, therapy resistance, high risk for distant metastasis and poor survival34, 35. As expected, Zeb1 transcript levels were increased during a TGFβ-induced EMT of different murine and human cellular systems (Supplementary Fig. 4a). Furthermore, small-interfering RNA-mediated knockdown of Zeb1 maintained an epithelial cell morphology in NMuMG/E9 and MCF10A cells induced to undergo an EMT and significantly reduced the migratory properties of mesenchymal (>20 days TGFβ) Py2T and 4T1 cells in trans-well Boyden chamber assays (Supplementary Fig. 4b–e).

We next examined whether miR-1199-5p indeed regulated Zeb1 levels during an EMT. Ectopic expression of miR-1199-5p in NMuMG/E9 and Py2T cells induced to undergo an EMT resulted in the stabilization of cell junction protein E-cadherin, while nuclear levels of Zeb1 were reduced (Fig. 4b, Supplementary Fig. 4f). Immunoblotting analyses for Zeb1 confirmed the repression of Zeb1 expression by miR-1199-5p in these murine cells (Fig. 4c, Supplementary Fig. 4g). Even though the seed match of human miR-1199-5p is not perfectly complementary to the seed sequence in the Zeb1 3′ UTR (mouse and human miR-1199-5p seed matches differ in one base), forced expression of hsa-miR-1199-5p still significantly reduced Zeb1 mRNA (Supplementary Fig. 3f) and protein (Supplementary Fig. 4h) levels in human MCF10A cells. These results further imply that even imperfect binding of a miRNA to its target RNA can efficiently downregulate its expression.

To validate the direct regulation of Zeb1 by miR-1199-5p on the post-transcriptional level during an EMT we made use of two Zeb1 3′ UTR luciferase reporter constructs, one containing the species conserved, wild-type miR-1199-5p seed sequence and the other one carrying a mutated version of the seed sequence with five nucleotides exchanged (Fig. 4a, d). Transient transfection of a miR-1199-5p mimic and the Zeb1 3′ UTR wild-type reporters in NMuMG/E9 cells revealed a significant decrease in luminescence, which was not observed with the mutant version of the reporter (Fig. 4d). Together, these data identify the key EMT TF Zeb1 as a direct target of miR-1199-5p, which thus represses Zeb1 expression at the post-transcriptional level and prevents an EMT.

Zeb1 directly controls the expression of miR-1199-5p

An important mechanism that appears to be responsible for EMT cell plasticity are double-negative feedback loops between miRNAs and key EMT TFs, functioning as molecular switches for various cell differentiation states18,19,20, 22, 36, 37. Because miR-1199-5p regulates the expression of Zeb1, we assessed whether Zeb1 would in turn regulate the expression of miR-1199-5p during an EMT. siRNA-mediated ablation of Zeb1 expression in NMuMG/E9 cells cultured in the presence of TGFβ for 4 days and transfected with either a miR-1199-promoter luciferase-reporter (pGL4 miR-1199 promoter) or a control promoter luciferase-reporter (pGL4; Supplementary Fig. 3b) revealed a significant increase in miR-1199 promoter activity upon loss of Zeb1 (Fig. 5a, left). Conversely, transient overexpression of Myc-tagged Zeb1 in epithelial NMuMG/E9 cells significantly decreased miR-1199 promoter activity (Fig. 5a, right). Furthermore, the regulation of miR-1199 promoter activity by loss or gain of function experiments of Zeb1 also correlated with an increase or decrease in endogenous miR-1199-5p transcript levels, respectively (Fig. 5b).

Fig. 5 Zeb1 directly regulates miR-1199-5p expression. a Zeb1 controls the promoter activity of the miR-1199 gene. NMuMG/E9 cells were transfected with a miR-1199 promoter Firefly luciferase reporter construct, a Renilla luciferase reporter construct as well as with (left) a siRNA against Zeb1 (siZeb1) or a negative control siRNA (siCtr) or (right) a Zeb1 expression construct (6xMyc-tag-Zeb1) or a negative control construct (6xMyc-tag). Cells transfected with siRNAs were cultured for 4 days with TGFβ, while cells transfected with 6xMyc or 6xMyc-tagged Zeb1 were cultured in the absence of TGFβ. Relative luminescence (Firefly/Renilla) was calculated and normalized to the control Firefly luciferase reporter (pGL4; mean fold changes±s.e.m.; n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05, **P < 0.01). b Zeb1 controls miR-1199-5p transcript levels. NMuMG/E9 cells were transfected with siRNAs (left) and Zeb1 constructs (right) as described in a and further cultured in the absence or presence of TGFβ. MiR-1199-5p transcript levels were examined by RT-PCR analysis (mean fold changes±s.e.m.; left: n = 5; right: n = 3). c Schematic presentation of the genomic localization of the murine miR-1199 gene and its promoter region. Red: mmu-miR-1199; grey: 2210011C24Rik gene; green: E-boxes (CANNTG, N = G or C); blue: promoter fragments examined by ChIP-qPCR analysis. d Zeb1 directly binds to the miR-1199 promoter. Chromatin of Py2T cells cultured in the absence (green) and presence of TGFβ (red) was subjected to chromatin immunoprecipitation with antibodies against Zeb1 followed by RT-PCR analysis using primers amplifying different regions of the miR-1199 promoter illustrated in c. An intergenic region was used as negative control. Data were normalized to control IgG and are presented as mean fold enrichment above background±s.e.m. (n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05, **P < 0.01, ****P < 0.0001). e Analysis of E-box-binding motifs in the miR-1199 promoter as potential Zeb1-binding sites. NMuMG/E9 cells were transfected and cultured as described in a (right). Relative luminescence (Firefly/Renilla) was calculated as described in a (mean fold changes±s.e.m.; n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05, ***P < 0.001) Full size image

The gene encoding for miR-1199-5p is located within the first CDS of an unknown, protein-coding gene (2210011C24Rik) on chromosome 8 in the mouse genome. Its localization is conserved in the human genome, where it is located in the first CDS of LOC113230, an orthologue of 2210011C24Rik, on chromosome 19 (Supplementary Fig. 5a). Notably, the transcript levels of the murine host gene are significantly downregulated during a TGFβ-induced EMT in different cellular models, as it was observed for miR-1199-5p (Supplementary Fig. 5b; Fig. 2a). Furthermore, gain and loss of function studies by siRNA-mediated ablation or transient overexpression of Zeb1 in NMuMG/E9 cells led to an increase or decrease in 2210011C24Rik expression, respectively (Supplementary Fig. 5c, d), indicating a co-regulation of miR-1199-5p and its host gene.

The murine miR-1199/2210011C24Rik promoter region encompasses several E-box motifs (CANNTG; N = G or C) as potential Zeb1-binding sites (Fig. 5c). We next performed chromatin immunoprecipitation (ChIP) for endogenous Zeb1 in epithelial Py2T cells and in 4 days TGFβ-treated Py2T cells, a time point displaying a robust increase in Zeb1 expression (Supplementary Fig. 4a), followed by quantitative RT-PCR analysis with various primer pairs covering different regions of the miR-1199/2210011C24Rik promoter. These experiments confirmed a direct binding of Zeb1 to a region with the closest proximity (+51/−52 bps) to the transcriptional start site (TSS) (Fig. 5d). Notably, Zeb1 binding was only observed in cells undergoing an EMT and not in their epithelial counterparts (Fig. 5d). Using the miR-1199/2210011C24Rik promoter luciferase reporter (Supplementary Fig. 3b), we individually mutated each of four Zeb1 E-box-binding sites (CAGGTG to CATTTG). Only mutation of E-box 1 (−18 bp from TSS) significantly ablated Zeb1-mediated repression of luciferase activity in NMuMG/E9 cells (Fig. 5e).

Together, the results show that Zeb1 is a direct transcriptional repressor of the miR-1199 gene. Hence, Zeb1 and miR-1199-5p act in a reciprocal fashion to repress each other.

Comparing miR-1199-5p and miR-200s function during an EMT

MiR-1199-5p’s function during an EMT as well as its regulation by the EMT TF Zeb1 resembles the activities of members of the miR-200 family, two of which, miR-200b-3p and miR-429-3p, have been identified by our functional EMT screen (Fig. 1)14,15,16,17.

Comparable to miR-1199-5p, miR-200b-3p and miR-429-3p transcript levels are also strongly reduced during a TGFβ-induced EMT in NMuMG/E9 cells (Supplementary Fig. 6a, Fig. 2a). The forced expression of miR-200b-3p or miR-429-3p in human MCF10A (Supplementary Fig. 6b, top) and murine NMuMG/E9 cells (Supplementary Fig. 1a–c) also prevented an EMT and maintained an epithelial morphology in the presence of TGFβ. However, similar to miR-1199-5p, expression of miR-200b-3p and miR-429-3p mimics in tumorigenic Py2T cells failed to completely block mesenchymal cell morphology, even though the mRNA levels of E-cadherin and Zeb1 were significantly increased or decreased, respectively (Supplementary Fig. 6b, c). Also comparable to miR-1199-5p, ectopic expression of miR-200b-3p or miR-429-3p significantly reduced cell migration and invasion of mesenchymal Py2T and 4T1 cells (Supplementary Fig. 2, Supplementary Fig. 6d). Notably, all three miRNAs are mechanistically embedded in a double-negative feedback regulation with Zeb118, 19 (Fig. 4, Fig. 5, Supplementary Fig. 6e).

Since miR-200b-3p, miR-429-3p and miR-1199-5p induced comparable functional outputs with regard to an EMT process, we assessed their effects on tumour progression in vivo. Stable expression of miR-1199, miR-200b (which also induced the expression of miR-429 and slightly miR-1199; Supplementary Fig. 6f) and miR-429 in metastatic 4T1 cells tagged by ZsGreen led to a reduction in Boyden chamber trans-well cell migration compared to an empty vector control (Supplementary Fig. 6g) and confirmed our results for their transient overexpression (Fig. 2g, Supplementary Fig. 6d).

Upon orthotopic transplantation of 4T1 cells into the mammary fat pads of immunodeficient NSG and NMRI mice, the forced expression of miR-1199, miR-429 or miR-200b induced a significant reduction in primary tumour growth over time (Fig. 6a). Notably, the high potential of 4T1 cells to metastasize to the lungs was reduced by the expression of miR-1199 and miR-429, however not by the expression of miR-200b (Fig. 6b–d). The failure of miR-200b to repress 4T1 metastasis was surprising, yet has been observed by others as well (G.J. Goodall, personal communication of unpublished data). Examining the metastatic outgrowth of 4T1 cells in the lung, expression of each of the three miRNAs significantly increased the size of the few metastatic nodules compared to the many nodules observed with empty vector-transduced cells (Fig. 6e). Therefore, the repressive growth effect observed for miR-1199, miR-200b and miR-429 in the primary tumour was not maintained in the outgrowth of tumour cells at the metastatic site. Of note, the number of circulating tumour cells isolated from the blood of tumour-bearing mice was decreased by the forced expression of miR-1199 and miR-429, but not by the expression of miR-200b (Fig. 6f, g).

Fig. 6 miR-1199 and miR-200s repress 4T1 murine breast cancer primary tumour growth and metastasis. a ZsGreen-labelled 4T1 cells stably expressing miR-1199, miR-200b or miR-429 were injected into the mammary fat pad of female NSG or NMRI mice as indicated, and primary tumour growth was analysed over time. Statistical significance compared to empty vector control: 7 days: miR-1199*, miR-429*; 11 days: miR-1199**, miR-429**, miR-200b**; 14 days: miR-1199***, miR-429**, miR-200b**; 18 days: miR-1199***, miR-429***, miR-200b*; 21 days: miR-1199***, miR-200b*. b Representative bright-field images of whole lungs (left), enlargement of indicated lung areas (middle) and H&E staining of lung sections (right) isolated from mice killed 21 days post cell injection. Scale bar, 1 mm. c, d The number of lung metastases per mouse was quantified microscopically by H&E staining. e The tissue area of all lung nodules was analysed and quantified microscopically by H&E staining. f, g CTCs isolated from the blood of tumour-bearing mice were isolated 21 days post cell injection and cultured for 5 days ex vivo. Cell colonies were visualized by MTT staining, imaged (f) and quantified (g) as indicated. NSG: 8 mice per group±s.e.m.; NMRI: 7–8 mice per group±s.e.m.; statistical analyses: Mann–Whitney U test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Full size image

In summary, miR-1199 and miR-429, but not miR-200b, are sufficient to reduce tumour cell intravasation into the blood circulation and the seeding of lung metastases. However, all three miRNAs seem to promote metastatic outgrowth once tumour cells have seeded in the lung parenchyma.

Common and distinct targets of miR-200s and miR-1199-5p

The functional similarities between miR-1199-5p and miR-200 family members observed during an EMT in vitro as well as during tumour progression in vivo begs the question about the shared and the distinct functions of miR-200 family members and miR-1199-5p in regulating an EMT. To reveal the transcriptomic effects of the various miRNAs during a TGFβ-induced EMT, we performed RNA sequencing of NMuMG/E9 cells transfected with miRNA mimics for miR-200b-3p, miR-429-3p and miR-1199-5p. As expected, miR-200b-3p or miR-429-3p mimics induced a block in EMT of NMuMG/E9 cells and led to an overall anti-correlative gene expression profile compared to mesenchymal, miR-Ctr-transfected cells (r = −0.505 and r = −0.51, respectively; Fig. 7a), similar to the profile observed with miR-1199-5p mimic (r = −0.324; Fig. 3a). Differential gene expression analysis (log2FC(±1); FDR <0.05) revealed 1097 and 1058 genes affected by miR-200b-3p and miR-429-3p, respectively, during an EMT, of which 982 genes were shared by the two miRNAs (Fig. 7a, b). Since they belong to the same miRNA family, such a high number of co-regulated genes can be explained by an identical seed sequence on their target mRNAs. However, the difference in the effects of the two miRNAs on lung metastasis is surprising (Fig. 6b–d), and we can only speculate whether the small pool of individual target genes for each of the miRNAs is the reason for the different outcomes in vivo.

Fig. 7 Shared and distinct target genes of miR-1199-5p, miR-200b-3p and miR-429-3p. a Overall gene expression regulation by miR-200b-3p and miR-429-3p during a TGFβ-induced EMT. RNA-sequencing analyses were performed on NMuMG/E9 cells transiently transfected with a miR-200b-3p, miR-429-3p or a negative control (miR-Ctr) mimic. Cells were then cultured in the presence or absence of TGFβ (0 day vs. 4 days). RNA sequencing and data analysis was performed as described for miR-1199-5p in Fig. 3a. Scatterplots depict the overall gene expression (log2FC) (anti-)correlation between miR-200b-3p 4 days vs. miR-Ctr 4 days (left) or miR-429-3p 4 days vs. miR-Ctr 4 days (right) over miR-Ctr 4 days vs. miR-Ctr 0 day. Differential expression analysis (red dashed line: log2FC(±1); FDR <0.05) identified 1097 genes and 1058 genes regulated during an EMT by miR-200b-3p and miR-429-3p, respectively. b The Venn diagram summarizes the number of genes commonly and individually regulated by miR-1199-5p (blue), miR-200b-3p (red) and miR-429-3p (green) during a TGFβ-induced EMT (data from Fig. 7a, Fig. 3a). c Functional annotation clustering analysis by DAVID of genes commonly regulated by miR-1199-5p, miR-200b-3p and miR-429-3p during an EMT. Presented are the top five biological processes, pathways (KEGG) and their associated P and Benjamini–Hochberg values. d The Venn diagram presents the number of commonly and individually regulated, predicted target genes of miR-1199-5p (blue), miR-200b-3p (red) and miR-429-3p (green) during an EMT. MirWalk2.0 was used to predict miRNA target genes that show reduced transcript levels upon forced expression of miR-1199-5p, miR-200b-3p and miR-429-3p during an EMT. e Shown are the mRNA levels of genes commonly repressed by miR-1199-5p, miR-200b-3p and miR-429-3p during an EMT (RNA-sequencing data: log2FC compared to miR-Ctr-transfected cells). f Schematic representation of the double-negative feedback loops between miR-200 family members and Zeb1 and between miR-1199-5p and Zeb1. Only six target genes are shared between miR-200 and miR-1199-5p among which is Zeb1. Each of them has individual target genes as indicated and thus may affect distinct biological functions Full size image

Directly compared to each other, miR-200b-3p, miR-429-3p and miR-1199-5p share 465 regulated genes during an EMT, which is more than half of the 787 genes regulated by miR-1199-5p alone (Fig. 7b, Fig. 3a). Functional annotation analysis revealed that these genes control the same biological processes and pathways as miR-1199-5p alone, that is, cell adhesion, ECM-receptor interactions and focal adhesions (Fig. 7c, Fig. 3b). A total of 267 genes are exclusively regulated by miR-1199-5p during an EMT.

Target prediction analysis by miRWalk2.030 for miR-200b-3p and miR-429-3p together with the differential expression analysis presented in Fig. 7a revealed 54 direct target genes commonly regulated by both miR-200 family members during an EMT (Fig. 7d, Supplementary Table 2), among them the key EMT TFs Snail1, Zeb1 and Zeb23. Six out of the 54 genes were also directly controlled by miR-1199-5p (Fig. 7d). Besides the common target Zeb1, mRNA levels for Ncs1, Cdon, Sox12, Zfp9, Col5a1 were also decreased by all three miRNAs during an EMT (Fig. 7e). Of note, 58 target genes were uniquely regulated by miR-1199-5p.

The results show that miR-1199-5p, miR-200b-3p and miR-429-3p tightly control TGFβ-induced EMT plasticity with similar potency, however, they only share the regulation of six gene transcripts, one of which is the critical EMT TF Zeb1. Yet, each of the miRNAs seems to have distinct functions by repressing a larger number of unique target mRNAs.