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• W2123011109 abstract "Article10 September 2009Open Access Myc-regulated microRNAs attenuate embryonic stem cell differentiation Chin-Hsing Lin Chin-Hsing Lin Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Aimee L Jackson Aimee L Jackson Department of Biology, Rosetta Inpharmatics LLC, Seattle, WA, USAPresent address: Regulus Therapeutics, 1896 Rutherford Rd, Carlsbad, CA 92008, USA Search for more papers by this author Jie Guo Jie Guo Department of Biology, Rosetta Inpharmatics LLC, Seattle, WA, USA Search for more papers by this author Peter S Linsley Peter S Linsley Department of Biology, Rosetta Inpharmatics LLC, Seattle, WA, USAPresent address: Regulus Therapeutics, 1896 Rutherford Rd, Carlsbad, CA 92008, USA Search for more papers by this author Robert N Eisenman Corresponding Author Robert N Eisenman Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Chin-Hsing Lin Chin-Hsing Lin Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Aimee L Jackson Aimee L Jackson Department of Biology, Rosetta Inpharmatics LLC, Seattle, WA, USAPresent address: Regulus Therapeutics, 1896 Rutherford Rd, Carlsbad, CA 92008, USA Search for more papers by this author Jie Guo Jie Guo Department of Biology, Rosetta Inpharmatics LLC, Seattle, WA, USA Search for more papers by this author Peter S Linsley Peter S Linsley Department of Biology, Rosetta Inpharmatics LLC, Seattle, WA, USAPresent address: Regulus Therapeutics, 1896 Rutherford Rd, Carlsbad, CA 92008, USA Search for more papers by this author Robert N Eisenman Corresponding Author Robert N Eisenman Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA Search for more papers by this author Author Information Chin-Hsing Lin1, Aimee L Jackson2, Jie Guo2, Peter S Linsley2 and Robert N Eisenman 1 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 2Department of Biology, Rosetta Inpharmatics LLC, Seattle, WA, USA *Corresponding author. Division of Basic Sciences, Fred Hutchinson Cancer Research Center A2M-025, 1100 Fairview Avenue N., POB 19024, Seattle, WA 98109-4417, USA. Tel.: +1 206 667 4445; Fax: +1 206 667 6522; E-mail: [email protected] The EMBO Journal (2009)28:3157-3170https://doi.org/10.1038/emboj.2009.254 There is a Have you seen ...? (October 2009) associated with this Article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Myc proteins are known to have an important function in stem cell maintenance. As Myc has been shown earlier to regulate microRNAs (miRNAs) involved in proliferation, we sought to determine whether c-Myc also affects embryonic stem (ES) cell maintenance and differentiation through miRNAs. Using a quantitative primer-extension PCR assay we identified miRNAs, including, miR-141, miR-200, and miR-429 whose expression is regulated by c-Myc in ES cells, but not in the differentiated and tumourigenic derivatives of ES cells. Chromatin immunoprecipitation analyses indicate that in ES cells c-Myc binds proximal to genomic regions encoding the induced miRNAs. We used expression profiling and seed homology to identify genes specifically downregulated both by these miRNAs and by c-Myc. We further show that the introduction of c-Myc-induced miRNAs into murine ES cells significantly attenuates the downregulation of pluripotency markers on induction of differentiation after withdrawal of the ES cell maintenance factor LIF. In contrast, knockdown of the endogenous miRNAs accelerate differentiation. Our data show that in ES cells c-Myc acts, in part, through a subset of miRNAs to attenuate differentiation. Introduction MicroRNAs (miRNAs), 21–23 nucleotide non-protein coding RNAs, act as powerful regulators of gene expression at the post-transcriptional level by targeting specific mRNA degradation or by suppression or activation of translation (Carrington and Ambros, 2003; Dykxhoorn et al, 2003; Grewal and Moazed, 2003; Pickford and Cogoni, 2003; Vasudevan et al, 2007). Studies from many laboratories have showed that miRNAs influence a wide range of biological processes including development, lifespan, metabolism, and cancer (Kato and Slack, 2008; Stefani and Slack, 2008). Regulation of miRNA genesis occurs at multiple levels in response to differentiation and mitogenic signalling (Thomson et al, 2006; Viswanathan et al, 2008). Some miRNAs are characteristic of specific differentiated cell types, whereas others are specifically expressed in stem and progenitor cells during early development (Lim et al, 2003; Chen et al, 2004; Poy et al, 2004; Chang and Mendell, 2007; Hwang and Mendell, 2007; Ibarra et al, 2007). Recent evidence has shown that miRNA mutations or deregulations correlate with different human cancers, and has also shown that miRNAs can function as tumour suppressors or oncogenes (Calin and Croce, 2006; Esquela-Kerscher and Slack, 2006; Medina and Slack, 2008; Tavazoie et al, 2008). The most well-characterized miRNA cluster involved in tumourigenesis, miR-17-92, was found to be induced by Myc oncoprotein expression (He et al, 2005; Mendell, 2005; O'Donnell et al, 2005). The Myc protein family comprises basic helix-loop-helix-zipper (bHLHZ) transcription factors (c-, N-, and L-Myc) that can each form obligate heterodimers with the small bHLHZ protein Max. Myc–Max heterodimers bind the E-box sequence CACGTG in many different cell types and activate transcription of a large number of genes, many of which are associated with cell growth. In addition, Myc has been implicated in transcriptional repression of many genes that normally limit cell-cycle progression (Adhikary and Eilers, 2005; Cole and Nikiforov, 2006; Kleine-Kohlbrecher et al, 2006). Myc's broad effects on both normal and abnormal cell behaviour have been generally assumed to relate to its regulation of RNA polymerase II transcription of protein coding target genes as well as RNA polymerase I and RNA polymerase III transcription of RNAs involved in translation and growth (Gomez-Roman et al, 2003; Arabi et al, 2005; Grandori et al, 2005). However, the demonstration that Myc also controls expression of a subset of miRNAs has added another class of critical Myc targets. Induced expression of c-Myc in the P493-6 B cell line showed upregulation of six miRNAs within the miR-17 cluster on chromosome 13 through direct binding of Myc to the miR-17 locus (He et al, 2005; O'Donnell et al, 2005). Recently, Myc was shown to be involved in repression of numerous miRNAs in tumour cell lines, including the let-7 tumour suppressor, which regulates expression of c-myc itself (Sampson et al, 2007; Chang et al, 2008) and miR-23a/b resulting in increased glutamine catabolism (Gao et al, 2009). Thus, Myc proteins seem to be intimately involved in the regulation of a broad range of miRNAs, many of which are likely to have key roles in cell proliferation and cancer (for reviews see Lotterman et al, 2008; Medina and Slack, 2008). Another biological setting in which Myc and miRNA regulation may converge is in stem cell self-renewal and pluripotency. Genetic studies in mice indicate that c-Myc is involved in the growth, proliferation, and differentiation of epidermal, neural and lung stem/progenitor cells, and haematopoietic stem cells (Arnold and Watt, 2001; Knoepfler et al, 2002; Okubo et al, 2005; Dubois et al, 2008). In embryonic stem (ES) cells c-Myc has been shown to be required for the maintenance of self-renewal whereas Myc downregulation on withdrawal of leukaemia inhibitory factor (LIF) is critical for differentiation (Cartwright et al, 2005). Furthermore, myc family genes, together with Oct4, Klf4, and Sox2, act to reprogramme differentiated cells into induced pluripotent stem (iPS) cells with ES cell properties, suggesting that c-Myc is a driver of pluripotency (Takahashi et al, 2007; Wernig et al, 2007). Although iPS conversion can occur without introduction of Myc, the overall yield of converted cells is dramatically decreased, as is the rate of conversion (Nakagawa et al, 2008). miRNAs have also been implicated in the ES cell function. Targeted deletion of the Dicer pre-miRNA processing ribonuclease abrogated ES cell differentiation whereas self-renewal was less drastically reduced (Kanellopoulou et al, 2005; Murchison et al, 2005). Indeed, a characteristic miRNA profile has been defined in murine and human ES cell lines, which overlaps with miRNAs implicated earlier in proliferation and tumourigenesis (Suh et al, 2004; Calabrese et al, 2007). Moreover, two studies have shown that pluripotency-related transcription factors regulate many miRNAs and are themselves subject to miRNA regulation (Marson et al, 2008; Tay et al, 2008). Furthermore, recent reports show that ES cell-specific miRNAs belonging to the miR-290 family promote the G1-S transition and self-renewal in ES cells as well as induced pluripotency during reprogramming of fibroblasts (Wang et al, 2008; Judson et al, 2009). These findings prompted us to determine whether c-Myc might contribute to ES cell pluripotency through regulation of miRNAs. Results Identification of c-Myc-induced miRNAs in ES cells To identify ES-specific c-Myc-induced miRNAs, we analysed three cell populations that permit us to compare ES cells to their differentiated and tumourigenic derivatives: (i) murine AK7 ES cells (ii) induced haematopoietic stem/progenitor cells (HSPs) derived from these ES cells on differentiation, and (iii) tumours from mice transplanted with ES cell-derived HSPs (Figure 1A). To determine the effects of increasing c-Myc levels, we used a c-myc-expressing lentiviral vector in ES cells (hereafter referred to as c-Myc+ cells) and identified c-Myc-induced miRNAs after normalization to a lentiviral vector control. We observed an 8–14-fold increase in c-myc RNA and protein levels as determined by qRT–PCR and immunoblotting, respectively (Supplementary Figure S1). We also generated differentiated cells from control and c-Myc+ ES cells. Treatment of murine ES cells with the cytokines IL-3, IL-6, and SCF resulted in induction of HSPs (Kushida et al, 2001; Burt et al, 2004). Finally, mixed T- and B-cell tumours were collected from irradiated mice transplanted with cytokine-induced c-Myc+ HSPs and compared with vector control transplanted HSPs in the miRNA profiling experiment (Supplementary Figure S2). Figure 1.miRNAs regulated by c-Myc in murine ES cells. (A) Scheme showing the experimental design for identification of miRNAs regulated by c-Myc in ES cells. c-Myc level was increased by lentiviral-delivered overexpression of c-Myc in ES cells (see Supplementary Figure S1). These ES cells, their differentiated haematopoietic stem/progenitor cell derivatives (HSP Myc+) and tumours after injection of HSP c-Myc+ cells into irradiated mice, were subjected to miRNA profiling. (B) Heatmap of miRNAs displaying significant regulation by c-Myc in ES cells and ES cell-derived differentiated cells or tumours. HSP: haematopoietic stem/progenitor cells derived by differentiation of ES cells; Tumours1–4: mixed T- and B-cell tumours derived by transplantation of Myc+ HSPs into irradiated mice. Note that results are shown for two HSP, and four tumour cell isolates independently derived from the same ES cell line. Bottom row shows miRNAs levels after c-Myc knockdown in WT ES cells. Inset: Fold change in miRNA copy number represents the ratio of miRNA level in ES cell in which c-Myc has been introduced (Myc+), or knocked down by c-myc shRNA (ES Myc KD), relative to lentiviral vector (see Supplementary Figure S1 for quantitation). Grey boxes represent no detectable signal. Download figure Download PowerPoint miRNA expression levels were measured using a quantitative primer-extension PCR assay as described earlier (Raymond et al, 2005; He et al, 2007). Primers representing 192 miRNAs were used in this study. To determine the effects of increasing c-Myc levels we calculated the fold change in miRNA copy number as the error-weighted average of the ratio in Myc+ cells relative to the lentiviral vector control (see Materials and methods). Figure 1B shows a heat map of the most significantly changed miRNAs in c-Myc+ versus vector controls in the ES cell-derived populations as described above. The figure displays results for two HSP, and four tumour cell isolates independently derived from the same ES cell line. Consistent with earlier findings (Mendell, 2005; O'Donnell et al, 2005), a subset of the oncogenic miRNA cluster, miR17-92, was highly induced in c-Myc+ differentiated cells, and in tumours from mice transplanted with c-Myc+ HSPs derived from our Myc+ ES cells (Figure 1B). However, the levels of these miRNAs were not substantially altered in c-Myc+ ES cells. We also identified four miRNAs, let-7, miR-29, miR-181, and miR-199 that are decreased in c-Myc+ ES cells relative to the vector control. Let-7 and miR-29 are downregulated by c-Myc in B-cell lymphomas (Chang et al, 2008). In addition, we find that miR-181A and miR-181B are sharply induced in Myc+ differentiated and tumour cells (Figure 1B). We also identified several miRNAs, miR-124, miR-135A, miR-135B, miR-141, miR-200, miR-302, miR-338, and miR-429 that seem to be upregulated specifically by c-Myc in ES cells (induced ∼four-fold in c-Myc+ cells; Figure 1B; see Supplementary Table S1 for complete list; Supplementary Figure S3A for northern blots). These miRNAs are induced by c-Myc in ES cells, but are either not increased or are strongly reduced in differentiated haematopoietic cells (Figure 1B; Supplementary Figure S3B) or in the tumours from transplanted mice. To further examine the regulation of these miRNAs by c-Myc we knocked down c-Myc levels two to four-fold in wild-type ES cells using lentiviral shRNA (Supplementary Figure S1B) and determined miRNA levels by quantitative primer-extension PCR as described. Our results show that after Myc knockdown the levels of miR-124, miR-135A, miR-135B, miR-141, miR-200, miR-302, miR-338, and miR-429 are sharply reduced (Figure 1B, compare top and bottom rows). The c-Myc knockdown experiment also confirms that expression of the miR17-92 cluster is diminished in shMyc-treated ES cells compared to the Myc+ differentiated and tumour cells, whereas miRNAs repressed in Myc+ ES cells are strongly activated after c-Myc knockdown (Figure 1B). Together, our data confirm that Myc activates or represses multiple miRNAs and identifies a subset of those whose specific expression in ES cells is Myc dependent. Earlier studies had reported that among the group of c-Myc-regulated miRNAs in ES cells described above, miR-135 and miR-124 regulate neurogenesis during central nervous system (CNS) development (Cao et al, 2007; Visvanathan et al, 2007). miR-302 is related to the miR-290 family several of whom have been recently shown to promote ES cell proliferation by targeting cell-cycle regulators (Wang et al, 2008). Interestingly, miR-291-3p, -294, and -295 seem to be directly regulated by Myc during reprogramming of fibroblasts to induce pluripotency (Judson et al, 2009). However, the targets and functions of several c-Myc-induced miRNAs, such as miR-141, miR-200, miR-338, and miR-429 in ES cells, have not been established. These miRNAs are endogenously expressed in ES cells in a Myc-dependent manner and their levels are augmented on c-Myc overexpression (Figure 1B; Supplementary Figure S3A and B). We therefore chose to focus on identifying targets and functions of these miRNAs. Analysis of c-Myc binding to selected miRNA loci To address whether c-Myc might regulate these miRNAs by direct binding to their genomic loci, we mapped the genomic-binding sites of c-Myc in ES cells using DNA derived from anti-c-Myc chromatin immunoprecipitation (ChIP) to probe a custom-designed DNA microarray (chip) tiling 3 Kb regions of miR-124, miR-135, miR-141, miR-200, miR-302, miR-338, and miR-429. Three independent sets of ChIPs with c-Myc antibody versus input DNA were hybridized to the custom array. The enrichment ratio for ChIP/input DNA (Figure 2A, grey dots, plotted on a log2 scale in which dotted purple line indicates log2=1) and the statistics program Algorithm for Capturing Microarray Enrichment (ACME) (Scacheri et al, 2006b) were used to identify chromosomal regions with statistically significant c-Myc binding (Figure 2A, red lines, see Material and methods section for details). Figure 2 shows binding by endogenous c-Myc proximal (within 100–200 bp) to coding regions for miR141, miR200/miR429, miR338. When we applied the ChIP–chip analysis to c-Myc+ ES cells we detected significant binding and/or additional binding sites at the miR-141, miR-200, miR-338 loci (Figure 2A). We also observed little or no endogenous c-Myc binding to either miR124 or miR135a whereas increased Myc levels generates binding in the vicinity of these two miR transcripts (Supplementary Figure S4). Interestingly, neither endogenous nor overexpressed c-Myc binds to the miR302 locus (Supplementary Figure S4), suggesting c-Myc indirectly induces miR302. We further validated the binding of Myc to genomic loci proximal to miR-141, miR-200, miR-338, and miR429 by qChIP–PCR using primers to amplify the bound regions defined by our array analysis (Figure 2B). Myc binding to miRNA loci occurred to the same extent as endogenous binding to the Myc target gene HirA (Chen et al, 2008; Kim et al, 2008). Evx2 served as a negative control. In contrast to ES cells, we were unable to detect c-Myc binding to the same miRNA loci in HSPs (data not shown), consistent with our data showing that these miRNAs are not induced by Myc in these differentiated cells (Figure 1B; Supplementary Figure S3B). Figure 2.Association of c-Myc with genomic regions proximal to miRNA genes. (A) c-Myc genomic binding was assessed by ChIP–chip analysis. ACME statistical analysis (–log10 P-value scale on left side of each panel), plotted as red dots, indicates statistically significant enrichment. The ACME data are derived from the fold enrichment ratios (log2 scale on the right side of each panel) representing the ChIP enriched versus total input genomic DNA for all probes within the indicated genomic regions and plotted as grey dots. x axis represents the genomic region surrounding the miRNA loci with tiling oligonucleotide probes on a custom-designed tiling array. Positions of the mature miRNA transcripts are indicated by green or orange bars. Arrows indicate direction of transcription. (B) c-Myc binding at microRNA loci. qChIP–PCR was carried out as described in Materials and methods using AK7 mES cells. Shown is endogenous c-Myc binding to the known c-Myc target gene HirA as a positive control. The Evx2 locus was used as a negative control. Analysis of Myc binding to the indicated miRNA genomic loci was carried out using ChIP from wild-type ES cells (endogenous) as well as ES cells overexpressing c-Myc (overexpression). Equal amounts of anti-c-Myc ChIP DNA and total input DNA were used for quantitative PCR using SYBR Green detection with an ABI7900HT system. The sequences of all primers for qChIP–PCR were based on the Myc-binding sites (100–200 bp from microRNA coding regions) identified from ChIP–chip analysis and are available on request. Bar heights represent the Myc-bound DNA as a percentage of the total input DNA as determined from four independent sets of experiments. Download figure Download PowerPoint Analysis of the targets of ES-specific c-Myc-induced miRNAs RNAs targeted by miRNAs are generally degraded and can be identified through expression array analysis (Lim et al, 2005; Visvanathan et al, 2007; Liu et al, 2008; Ziegelbauer et al, 2009). Therefore, to assess the extent to which miR-141, miR-200a, miR-429, miR-338-3p, and miR-302a control gene expression in ES cells, we used expression array profiling to identify transcripts regulated by each miRNA. An RNA duplex was designed for each miRNA in which the guide (active) strand matches the mature form of the natural miRNA (see Supplementary Table S2 for sequences). This approach has been used extensively to identify and confirm miRNA targets (Lim et al, 2005; Visvanathan et al, 2007). Duplexes were transfected into ES cells, and RNA was collected 12 or 24 h later. The miRNA-regulated genes were identified using oligonucleotide microarrays representing 21 000 genes. Transcripts regulated by the miRNA were identified as those whose expression was significantly decreased with a P-value ⩽0.01 relative to mock-transfected cells (see Materials and methods). Two independent experiments were performed, one including both 12 and 24 h time points after transfection, and one analysing a 24 h time point. The 12 h sample was included as an early time point to show trends in kinetic regulation of transcripts and aid in identification of direct targets. Figure 3 shows a heat map for transcripts regulated by miR-141, miR-200, miR-302, miR-338, and miR-429. We observed good agreement among the transcripts regulated in the individual 24 h samples by miR-141 and miR-200. The duplexes corresponding to miR-141 and miR-200 had overlapping but non-identical seed regions (see Supplementary Table S2 for sequences). Consistent with this, the signatures for these miRNAs were partially overlapping, that is some transcripts were regulated in common by both miRNAs, whereas some transcripts were regulated only by the individual miRNAs. miR-429, with a seed region unrelated to miR-141 or miR-200, regulated an independent set of transcripts. The transcripts showing significant downregulation for each miRNA were enriched in 3′ UTR sequences complementary to the seed region of the transfected miRNA (Figure 3). The signatures for each miRNA target set showed enrichment for all three hexamers corresponding to the seed region of that miRNA. Hexamer enrichment ranged from an E-value (P-value with Bonferroni correction) of 10−2–10−13. Thus, the microarray analysis identified transcripts likely to be direct targets of the miRNAs. Similar data were obtained for miR-338 and miR-302 (see Supplementary Table S3). Figure 3.Expression profiling identifies putative targets of c-Myc-induced miRNAs. Heatmap of transcript levels after transfection of ES cells with miR-141, miR-200, miR-338, miR-302, or miR-429. ES cell RNA was hybridized to Agilent microarrays containing oligonucleotides corresponding to approximately 21 000 genes. Transcript regulation was calculated as the error-weighted mean log10 ratio for each transcript across the fluor-reversed pair. Inset shows colour scale indicating change in expression level. The white box(es) highlights transcripts downregulated (P-value <0.01 relative to mock-transfected cells) in common across time points and replicates (24 h time point) for the same miRNA. The seed region hexamers enriched in the signature are indicated for each miRNA (gold lettering). Hexamers are listed from top to bottom for each miRNA as hexamer 1 (positions 1–6), hexamer 2 (positions 2–7), and hexamer 3 (positions 3–8). Samples marked ‘a’ and ‘b’ represent independent experiments. The dendrogram at the top of the panel clusters transcripts with similar expression patterns. Download figure Download PowerPoint We reasoned that if the miRNAs identified here are part of a c-Myc-regulated pathway in ES cells, then the transcripts downregulated by the miRNAs should overlap with transcripts downregulated by c-Myc overexpression. We therefore looked for genes whose expression is downregulated by both miRNA transfection and in response to c-Myc overexpression (Figure 4). Myc-regulated genes in ES cells were identified using the 21 000 gene oligonucleotide microarray as described above. Approximately one-third of the targets for each miRNA overlapped with c-Myc-regulated transcripts (Figure 4). As a control we examined miR-382, an miRNA that has not been found to be regulated by c-Myc, and determined that only 2 of 65 miR-382 repressed targets overlap with genes downregulated by c-Myc, a number lacking statistical significance (P=0.12) (Figure 4). Figure 4.The intersection of putative targets of c-Myc-induced miRNAs and targets regulated by c-Myc. Venn diagrams illustrating the overlap among genes downregulated by c-Myc and by each miRNA tested. The numbers in the circles represent the number of genes significantly regulated (P<0.01) by c-Myc and by the miRNAs indicated. The hypergeometric probabilities for the overlaps are given below each Venn diagram. miR-382 is a control miRNA not induced by c-Myc in ES cells. Download figure Download PowerPoint Among the genes significantly repressed by both c-Myc and the c-Myc-induced miRNAs, a subset has been implicated in growth arrest and differentiation of a number of cell types (Supplementary Table S3; see Discussion). To validate the expression profiling results, we employed qRT–PCR to assess expression of 20 potential targets 24 h after transfection of duplex miRNAs into ES cells (Figure 5A). We confirmed by northern blotting that the levels of introduced miRNAs are comparable to those following induction by c-Myc (see Supplementary Figure S5). Our results show that introduction of these miRNAs into ES cells is associated with decreased transcript levels for the predicted miRNA targets. Furthermore, using immunoblotting we found that the protein levels of several target genes, such as amphiregulin (Areg), Neuropilin 1 (Nrp1), cadherin 11 (Cdh11), and TGFβ receptor 3 (Tgfbr3), were sharply decreased at 48 h after miRNA transfection (Figure 5B). Figure 5.Target gene specificity of Myc-induced miRNAs. (A) The transcript levels of the indicated microRNA targets were validated by real-time PCR. RNA extracted 24 h after transfection of miRNA duplexes into ES cells was used to determine expression of target genes by real-time PCR. Each bar represents the average ΔΔCt on a log2 scale of triplicate sets of experiments after normalization to internal controls (ΔCt) and mock control (ΔΔCt). Error bars show standard error of the mean. (B) Total protein extracted from ES cells 48 h post transfection of miRNA duplexes was analysed by immunoblotting with commercially available antibodies. Mock-transfected cells were used as controls and γ-tubulin as loading control. (C) microRNA target specificity determined by dual luciferase reporter assay. Sequences complementary to the seed region of miR-141, miR-200, and miR-429 were present in the 3′ UTRs of Cdh11, Nrp1, and zfh1, respectively. To disrupt base pairing with miRNAs we mutated the 3′ UTR complementary seed sequences from ‘AACACT’ to ‘ACAACT’ for the 3′ UTR of Cdh11, from ‘AACAC’ to ‘AGCGC’ for Nrp1, and from ‘TAATAC’ to ‘TCGTAC’ for Zfh1a. The wild type or mutant 3′ UTRs derived from the indicated target genes were linked to luciferase and transfected into HeLa cells together with the indicated miRNAs. The fold change represents the ratio of firefly luciferase (FL)/Renilla luciferase (RL). Error bars represent standard error from four independent experiments. **P-value=0.002–0.008; *P-value=0.03. Download figure Download PowerPoint To determine whether miRNA target downregulation is mediated through recognition of the miRNA seed sequences, we cloned 3′ UTRs from Cdh11, Nrp1, and the zinc-finger homeobox gene (Zfh1a), which are target genes of miR-141, miR-200, and miR-429, respectively (Figure 5C). These 3′ UTRs contain seed homology sequences specific for each miRNA. We next made point mutations by site-directed mutagenesis within the 3′ UTR seed homology elements to test the miRNA target site specificity. We generated reporter constructs containing the wild type and mutant target 3′ UTR sequences linked to luciferase. The wild type or mutant reporters were co-transfected with individual miRNA duplexes into HeLa cells. Data were normalized to a co-transfected Renilla luciferase (RL) control. Figure 5C shows that miR-141, miR-200, and miR-429 specifically inhibit activity from their cognate wildt-ype reporter genes whereas mutagenesis of the miRNA recognition sites abolishes the inhibition by these miRNAs. As a further test of specificity we determined the effect of each miRNA duplex on endogenous Cdh11, Nrp1, and Zfh1a RNA levels by qRT–PCR (Supplementary Figure S6). We compared miRNA duplexes with wild-type and scrambled seed regions (Supplementary Figure S6A). In each case, downregulation of the specific target gene for a given miRNA is dependent on the miRNA having an intact seed region homologous to the target gene 3′ UTR. We also used LNA-anti-miRNAs (see below) along with scrambled controls to examine target gene expression. As shown in Supplementary Figure S6B, upregulation of RNA expression of the endogenous target gene is dependent on a wild-type seed region. We further extended these experiments by examining target gene protein levels. After introduction of c-Myc into ES cells we observe decreased levels of Nrp1, Cdh11, and Areg proteins, as determined by immunoblotting (Supplementary Figure S6C). However, treatment of the c-Myc-overexpressing cells with LNA-anti-miRNAs, directed against the endogenous Myc-induced miRNAs that specifically target these gene products, results in upregulation of their protein levels (Supplementary Figure S6C). Taken together, our transient reporter assays and determination of endogenous target gene levels support the idea that regulation of the miRNA t" @default.
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• W2123011109 title "Myc-regulated microRNAs attenuate embryonic stem cell differentiation" @default.
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• W2123011109 doi "https://doi.org/10.1038/emboj.2009.254" @default.
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