microRNA-101 is a potent inhibitor of autophagy
2011; Springer Nature; Volume: 30; Issue: 22 Linguagem: Inglês
10.1038/emboj.2011.331
ISSN1460-2075
AutoresLisa B. Frankel, Jiayu Wen, Michael Lees, Maria Høyer-Hansen, Thomas Farkas, Anders Krogh, Marja Jäättelä, Anders H. Lund,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle13 September 2011free access microRNA-101 is a potent inhibitor of autophagy Lisa B Frankel Lisa B Frankel Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jiayu Wen Jiayu Wen Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Michael Lees Michael Lees Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Maria Høyer-Hansen Maria Høyer-Hansen Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark Search for more papers by this author Thomas Farkas Thomas Farkas Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark Search for more papers by this author Anders Krogh Anders Krogh Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Marja Jäättelä Marja Jäättelä Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark Search for more papers by this author Anders H Lund Corresponding Author Anders H Lund Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Lisa B Frankel Lisa B Frankel Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Jiayu Wen Jiayu Wen Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Michael Lees Michael Lees Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Maria Høyer-Hansen Maria Høyer-Hansen Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark Search for more papers by this author Thomas Farkas Thomas Farkas Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark Search for more papers by this author Anders Krogh Anders Krogh Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Department of Biology, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Marja Jäättelä Marja Jäättelä Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark Search for more papers by this author Anders H Lund Corresponding Author Anders H Lund Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark Search for more papers by this author Author Information Lisa B Frankel1, Jiayu Wen1,2, Michael Lees1, Maria Høyer-Hansen3, Thomas Farkas3, Anders Krogh1,2, Marja Jäättelä3 and Anders H Lund 1 1Biotech Research and Innovation Centre and Center for Epigenetics, University of Copenhagen, Copenhagen, Denmark 2Department of Biology, University of Copenhagen, Copenhagen, Denmark 3Apoptosis Department and Centre for Genotoxic Stress Research, Institute of Cancer Biology, Danish Cancer Society, Copenhagen, Denmark *Corresponding author. Biotech Research and Innovation Centre, University of Copenhagen, Ole Maaløes Vej 5, Copenhagen 2200, Denmark. Tel.: +45 35325657; Fax: +45 35325669; E-mail: [email protected] The EMBO Journal (2011)30:4628-4641https://doi.org/10.1038/emboj.2011.331 There is a Have you seen? (November 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Autophagy is an evolutionarily conserved mechanism of cellular self-digestion in which proteins and organelles are degraded through delivery to lysosomes. Defects in this process are implicated in numerous human diseases including cancer. To further elucidate regulatory mechanisms of autophagy, we performed a functional screen in search of microRNAs (miRNAs), which regulate the autophagic flux in breast cancer cells. In this study, we identified the tumour suppressive miRNA, miR-101, as a potent inhibitor of basal, etoposide- and rapamycin-induced autophagy. Through transcriptome profiling, we identified three novel miR-101 targets, STMN1, RAB5A and ATG4D. siRNA-mediated depletion of these genes phenocopied the effect of miR-101 overexpression, demonstrating their importance in autophagy regulation. Importantly, overexpression of STMN1 could partially rescue cells from miR-101-mediated inhibition of autophagy, indicating a functional importance for this target. Finally, we show that miR-101-mediated inhibition of autophagy can sensitize breast cancer cells to 4-hydroxytamoxifen (4-OHT)-mediated cell death. Collectively, these data establish a novel link between two highly important and rapidly growing research fields and present a new role for miR-101 as a key regulator of autophagy. Introduction Autophagy is a cellular self-catabolic degradation process in which cytoplasmic constituents are sequestered into double membrane vesicles called autophagosomes, and subsequently degraded via the lysosomal pathway (He and Klionsky, 2009). In addition to its important housekeeping and homeostatic functions at basal levels, autophagy promotes the survival of starved or stressed cells through the recycling of nutrients and through removal of damaged proteins and organelles (Mathew et al, 2007; Mizushima et al, 2008). Autophagy is essential for normal development (Yue et al, 2003; Kuma et al, 2004) and defects in this process are linked with numerous human diseases including neurodegenerative disorders and cancer (Levine and Kroemer, 2008; Chen and Debnath, 2010). In a tumour microenvironment, autophagy can promote cancer cell survival in response to metabolic stress (Degenhardt et al, 2006; Karantza-Wadsworth et al, 2007). However, progressive autophagy can also induce cell death and human cancers often display inactivating mutations in autophagy promoting genes (Corcelle et al, 2009; Chen and Karantza, 2011). Thus, in relation to tumourigenesis, the role of autophagy is complex and likely depends on the genetic composition of the cell as well as the environmental cues the cell is exposed to. miRNAs are small non-coding RNAs which post-transcriptionally regulate gene expression, predominantly through imperfect base pairing with the 3′-untranslated region (3′UTR) of target mRNAs (Valencia-Sanchez et al, 2006). miRNA-mediated repression of gene expression occurs through complex mechanisms which are not fully understood, including translational inhibition and mRNA degradation (Filipowicz et al, 2008). Substantial evidence implicates a functional role for miRNAs in different cancers and in support of this ∼50% of miRNAs are located at fragile sites of the genome, which are commonly amplified or deleted in human cancers (Calin et al, 2004). Accordingly, miRNA expression is often deregulated in cancer and miRNAs can act as oncogenes or tumour suppressors. In addition, miRNA expression profiling can be used to predict the clinical outcome of cancer patients (Lu et al, 2005; Volinia et al, 2006; Jiang et al, 2008). Despite recent advances in understanding the molecular mechanisms, which regulate autophagy, several gaps remain (Behrends et al, 2010; Lipinski et al, 2010; Szyniarowski et al, 2011). Considering the widespread implications of both miRNAs and autophagy in cancer-related processes and given the lack of current evidence linking these two rapidly growing fields of research, we were prompted to search for miRNAs which regulate autophagy. Through a luciferase-based functional screening approach, we identified miR-101 as a potent inhibitor of autophagic flux in MCF-7 breast cancer cells. miR-101 is lost in several cancer types including liver, prostate and breast, and emerging evidence suggests a tumour suppressive role for this miRNA (Varambally et al, 2008; Su et al, 2009; Chiang et al, 2010; Buechner et al, 2011). Accordingly, miR-101 induces apoptosis and suppresses tumourigenicity in vitro and in vivo, and was recently reported to inhibit migration and invasion of gastric cancer cells (Varambally et al, 2008; Su et al, 2009; Wang et al, 2010). Established targets for miR-101 include enhancer of zeste homologue 2 (EZH2), cyclooxygenase-2 (COX-2), amyloid precursor protein (APP) and myeloid cell leukaemia sequence-1 (MCL-1) (Varambally et al, 2008; Strillacci et al, 2009; Su et al, 2009; Vilardo et al, 2010). We report three novel miR-101 targets identified by microarray expression analysis, STMN1, RAB5A and ATG4D, which we show to be important autophagic regulators. Importantly, ectopic expression of a miR-101-resistant form of STMN1 alleviates miR-101-mediated repression of autophagy, confirming the functional importance of this target. Finally, in line with recent evidence suggesting that autophagic inhibition sensitizes breast cancer cells to cell death induced by various chemotherapeutic agents (Abedin et al, 2007; de Medina et al, 2009) we show that miR-101, likely through its inhibition of autophagy, can sensitize to 4-OHT treatment by enhancing 4-OHT-mediated cell death. Results A functional screen identifies miRNAs affecting autophagy To identify miRNAs regulating autophagy, we employed a functional screening approach using a cell-based assay system in which the autophagosomal marker, MAP1-LC3 (LC3), is fused to a renilla luciferase (RLuc) reporter molecule forming the RLuc–LC3 fusion protein (Farkas et al, 2009). As LC3 itself is specifically degraded by autophagy, the level of autophagy in an MCF-7 reporter cell line stably expressing wild-type RLuc–LC3 (RLuc–LC3WT) can be measured in real time using an in vivo RLuc substrate. As a reference control, MCF-7 cells expressing a mutant fusion protein, RLuc–LC3G120A, which is unable to undergo autophagosomal localization and is thereby not specifically degraded by autophagy, are assayed in parallel. The autophagic flux can hence be evaluated as the change in the relative levels of these two fusion proteins (hereafter denoted as LC3WT/LC3G120A; Farkas et al, 2009). The reporter cell system was transfected in 96-well format with a library of ∼470 miRNA precursor molecules covering the most abundant human miRNAs following the scheme outlined in Figure 1A. We measured the intrinsic effect of overexpressing the miRNAs on the basal autophagic flux at 42 h post-transfection after which etoposide was added. The autophagy-inducing effect of etoposide is well documented (Shimizu et al, 2004; Katayama et al, 2007; Farkas et al, 2009), and including etoposide treatment in the screen enabled greater sensitivity for the detection of miRNAs blocking autophagy. The RLuc activity was measured again at 12 and 24 h following etoposide addition. Aside from miRNAs, a number of control siRNAs were included in the screen as shown in Supplementary Figure S1. Knockdown of the essential autophagy component Beclin-1 (Supplementary Figure S1A) effectively inhibited autophagy as evident from measurements of the autophagic flux (Supplementary Figure S1B). Transfection efficiency throughout the screen was monitored using a siRNA against RLuc (Supplementary Figure S1C). Furthermore, scrambled control siRNAs scored similarly to the average of the entire miRNA library, ensuring that this negative control was suitable (Supplementary Figure S1D). To monitor and ensure reproducibility, the screening procedure was repeated three times. Figure 1.Screening approach for identification of miRNAs regulating autophagic flux in MCF-7 cells. (A) Outline of the timeline used for the screening assay. (B) Combined results from three independent screens, 66 h after transfection. MCF-7 RLuc–LC3WT and RLuc–LC3G120A were reverse transfected with the Ambion Pre-miR Library in three independent screens (see Materials and methods for details). The miRNAs were ranked according to log2 LC3WT/LC3G120A luciferase activity values (y axis). Highlighted in red, green and blue are three miRNAs, which consistently inhibited autophagic flux in the three independent screens (miR-145 P=0.0033, miR-95 P=0.0033 and miR-101 P=0.0305). Download figure Download PowerPoint Reasoning that autophagy could be induced as a stress response following overexpression of non-physiological levels of miRNAs or from miRNAs expressed outside their normal physiological context, we chose to focus on miRNAs inhibiting autophagy. Figure 1B shows the combined results of all three screens in which the miRNAs have been ranked according to fold change values (LC3WT/LC3G120A). Statistical analysis using a non-parametric rank product method based on ranks of fold changes (Breitling et al, 2004) revealed miR-95, miR-145 and miR-101 as the three most consistent, high-ranking miRNAs, which significantly inhibited autophagic flux in all three screens. miR-101 is regulated during autophagy Among the miRNAs identified to repress autophagy, miR-101 and miR-145 were immediately interesting due to well-established links to cancer (Varambally et al, 2008; Su et al, 2009; Kent et al, 2010). Since we have previously seen that miR-145 levels are undetectable in MCF-7 cells (Gregersen et al, 2010) we focused our attention on miR-101. To explore possible links between autophagy and miR-101 expression, we measured the level of endogenous miR-101 under basal growth conditions and following induction of autophagy. Detection of miR-101 in MCF-7 cells by quantitative PCR (qPCR) analysis revealed that endogenous miR-101 expression is increased by various triggers of autophagy including starvation, rapamycin and etoposide treatment (Supplementary Figure S2A and B; top). The mammalian target of rapamycin complex 1 (mTORC1) is a key negative regulator of autophagy signalling and its activation status reflects the level of autophagy in cells (Jung et al, 2010). Phospho-S6-kinase (p-S6K), a direct target of mTORC1, was used to indicate the extent of mTORC1 inactivation caused by these treatments (Supplementary Figure S2A and B; bottom). Considering the differential effects of etoposide and rapamycin, mTORC1-independent signalling could also influence the upregulation of this miRNA. These data indicate a potential physiological role for endogenous miR-101 in autophagy regulation in MCF-7 cells, prompting us to further analyse its function. Overexpression of miR-101 represses autophagy To validate the repressive effect of miR-101 overexpression on autophagy, three independent methods were employed. First, MCF-7 cells stably expressing an eGFP–LC3 fusion protein were used to quantify accumulation of autophagosomes by eGFP–LC3 translocation. As can be seen from Figure 2A, the percentage of eGFP–LC3 puncta-positive cells was significantly reduced in cells overexpressing miR-101, relative to cells transfected with the controls miR-203 and a siRNA against RLuc. miR-203 was chosen as a suitable miRNA-negative control since it had no effect on autophagy, behaving similarly to control siRNAs. Importantly, the effect of miR-101 was comparable to that of cells treated with a siRNA against Beclin-1. Representative images are shown in Figure 2B. Second, the effect of miR-101 on autophagic flux was confirmed using the RLuc assay from the screen. Also in this assay, miR-101 significantly inhibited the autophagic flux (both basal and etoposide induced) to an extent similar to Beclin-1 knockdown and significantly more than the controls (Figure 2C). Finally, we assayed the ability of miR-101 to affect the level of p62. The poly-ubiquitin binding protein p62 binds directly to LC3 and acts as a selective autophagy receptor and molecular carrier of cargo to be degraded by autophagy (Bjorkoy et al, 2005; Pankiv et al, 2007). As p62 itself localizes to autophagosomes and is degraded during autophagy, the level of p62 reflects the autophagic turnover. As evident from Figure 2D, overexpression of miR-101 in MCF-7 cells results in the accumulation of p62, reflecting an inhibition of autophagy. Hence, in accordance with the original screen, all three assays employed validate that overexpression of miR-101 inhibits autophagy in MCF-7 cells. Importantly, this finding extends to other cell lines including T47D, HEK and HeLa cells, as assessed by p62 expression levels, indicating that miR-101 regulation of autophagy is a general phenomenon (Supplementary Figure S3). Figure 2.miR-101 overexpression inhibits autophagy. (A) miR-101 overexpression inhibits eGFP–LC3 translocation. MCF-7 eGFP–LC3 cells were transfected with indicated miRNAs or siRNAs and fixed 72 h post-transfection. Percentage of eGFP–LC3 puncta-positive cells was quantified by automated image acquisition and analysis using a threshold of >5 dots/cell. Data are shown as the mean±s.d. of five replicates and is representative of three independent experiments. *P<0.05, **P<0.005. (B) Representative images from the quantification shown in (A). Scale bars represent 20 μM. (C) miR-101 overexpression inhibits autophagic flux. MCF-7 RLuc–LC3WT and RLuc–LC3G120A cells were reverse transfected with indicated miRNAs or siRNAs and 42 h later 50 μM etoposide was added. Luciferase activity was measured at 42, 54 and 66 h after transfection. Data are shown as the mean±s.d. of three replicates and are representative of three independent experiments. *P<0.05, **P 5 dots per cell. Data are shown as the mean±s.d. of five replicates and are representative of three independent experiments. **P<0.005. (B) Representative images from the quantification shown in (A). Scale bars represent 20 μM. (C) miR-101 inhibition induces autophagic flux. MCF-7 RLuc–LC3WT and RLuc–LC3G120A cells were reverse transfected with indicated LNAs or siRNAs and 42 h later 50 μM etoposide was added. Luciferase activity was measured at 42, 54 and 66 h after transfection. Data are shown as the mean±s.d. of three replicates and are representative of three independent experiments. **P<0.005. (D) miR-101 inhibition leads to decreased p62 expression. Western blot analysis 72 h after transfection with LNA miR-101 or LNA scramble. p62 bands were quantified relative to the vinculin loading control using ImageJ software and the relative quantifications are shown. The data are representative of three independent experiments. Download figure Download PowerPoint Experimental identification of miR-101 targets Having established a role for miR-101 in autophagy, we next wanted to clarify the underlying mechanism by identifying the direct downstream targets, which are repressed. We have previously used miRNA inhibition or overexpression coupled to transcriptome profiling to identify biological responses of—and direct targets for—several miRNAs (Frankel et al, 2008; Christoffersen et al, 2010; Gregersen et al, 2010). To identify targets of miR-101, MCF-7 cells were transfected with a miR-101 precursor or scramble control. Transfections were performed in triplicate and total RNA was harvested 24 h post-transfection and analysed using Affymetrix HG-U133 2.0 arrays. Following filtering, normalization and statistical analysis, we found 1107 differentially expressed transcripts of which 847 were downregulated and 260 were upregulated upon miR-101 overexpression (false discovery rate (FDR) ⩽0.1). Comparison of downregulated, upregulated and no change transcript sets revealed a highly significant enrichment for miR-101 seed site occurrence in the 3′UTRs of the downregulated transcripts for four different seed types (Figure 4A). Among the different seed types, the strongest enrichment was observed for the 8mer seed sites. To assess the efficacy of different seed types on mRNA repression, we compared the cumulative distribution functions (CDFs) of fold change for transcripts containing miR-101 seed sites or no seed sites (Figure 4B). The downregulation levels of transcripts with all seed match types were significantly higher than those without seed sites (P-values <0.0005). We validated the microarray study by qPCR analysis of 14 transcripts from the downregulation set, all of which were found to be downregulated in an independent experiment (Figure 4C). Supplementary Table I summarizes the complete list of 1107 differentially expressed transcripts. Figure 4.Identification of miR-101 targets by microarray analysis after miR-101 overexpression. (A) Seed site enrichment. The proportion and actual number of transcripts in the up, down and no change sets with different seeds types are shown. The seed types are mutually exclusive (see Materials and methods). Enrichment significance is labelled as ***P<0.0005 for down set versus up set and down set versus no change set. ‘⩾7mer’ includes 8mer, 7mer-m8 and 7mer-A1 sites. P-values for ⩾7mer seed site enrichment were 1.4e−16 (down versus up) and 1.9e−59 (down versus no change). (B) Different seed match types affect the level of transcript downregulation. Cumulative distribution functions for fold changes (log2FC) of transcripts containing different seed types or no seed sites for miR-101. X axis is log2FC from high downregulation to no change (log2FC=0) and from no change to high upregulation and y axis is the fraction of transcripts smaller than or equal to a certain fold change. For example, the solid grey vertical line drawn at log2FC=−0.2 approximately corresponds to 45% of transcripts containing 8mer seed type that were downregulated at least log2FC −0.2, compared with only 10% of transcripts containing 6mer seed type. (C) qPCR validation of 14 downregulated transcripts from an independent transfection experiment. For each transcript, the values are normalized relative to the scramble control sample and to the housekeeping gene. The error bars represent ±s.d. of three replicates. Download figure Download PowerPoint Knockdown of STMN1, RAB5A and ATG4D inhibits basal and rapamycin-induced autophagy In order to narrow down our search for biologically relevant miR-101 targets, we chose eight genes identified by the array analysis with potentially interesting links to autophagy and knocked them down individually by siRNA. We reasoned that knockdown of functionally important targets downstream of miR-101, should, at least in part, phenocopy the effect of overexpressing miR-101. We tested the effect of these siRNAs on eGFP–LC3 translocation and identified three genes, STMN1, RAB5A and ATG4D, which when knocked down inhibited both basal and rapamycin-induced autophagy (Figure 5A). The knockdown efficiencies of the individual siRNAs were measured by qPCR (Supplementary Figure S5B). Figure 5.siRNAs against STMN1, RAB5A and ATG4D inhibit basal and rapamycin-induced autophagy. (A) siRNA knockdown of a panel of genes selected from the array analysis led to identification of STMN1, RAB5A and ATG4D as potentially interesting miR-101 target candidates. MCF-7 eGFP–LC3 cells were transfected with indicated miRNAs or siRNAs and fixed 72 h post-transfection. Two hours prior to fixation, cells were treated with 200 nM rapamycin or left untreated. Percentage of eGFP–LC3 puncta-positive cells was quantified as described previously. Data are shown as the mean±s.d. of five replicates and are representative of two or three independent experiments. *P<0.05 relative to miR-203 untreated, #P<0.05 relative to miR-203 rapamycin, ##P<0.005 relative to miR-203 rapamycin. (B) Knockdown of STMN1, RAB5A and ATG4D decreases autophagic flux. MCF-7 RLuc–LC3WT and RLuc–LC3G120A cells were reverse transfected with indicated miRNAs or siRNAs and 42 h later luciferase activity was measured. Data are shown as the mean±s.d. of three replicates and are representative of at least three independent experiments. **P<0.005, ***P<0.0005. Download figure Download PowerPoint STMN1 encodes Stathmin/Oncoprotein18, a cytosolic phosphoprotein that regulates microtubule dynamics and is found overexpressed in many cancers (Marklund et al, 1996; Rana et al, 2008). In an independent siRNA-based screen, we had previously identified Stathmin as a regulator of autophagy (Maria Høyer-Hansen and Marja Jäättelä, unpublished data). RAB5A, a small GTPase that regulates early endocytosis, was previously suggested to act at an early stage of autophagosome formation (Ravikumar et al, 2008). Finally, ATG4D is a member of the ATG4 family of cysteine proteases, which regulate autophagosome biogenesis through the processing of LC3, allowing its subsequent conjugation to phosphatidylethanolamine (PE) on autophagosomal membranes (Marino et al, 2003; Scherz-Shouval et al, 2007). While overexpression of miR-101 resulted in ∼50% reduction in transcript abundance for STMN1, RAB5A and ATG4D (Figure 4C), specific siRNAs reduced the levels to varying degrees (∼45% for RAB5A, ∼25% for ATG4D and <5% for STMN1; Supplementary Figure S5B). In addition to regulating steady-state autophagy, knockdown of these three genes partly abrogated the autophagic flux, as shown by a significant increase in the LC3WT/LC3G120A ratio (Figure 5B). In both assays, the extent of autophagic inhibition by these siRNAs was similar to Beclin-1 knockdown or miR-101 overexpression (Figure 5A and B). Since the downregulation of STMN1, RAB5A and ATG4D also decreased eGFP–LC3 translocation in the face of Rapamycin treatment (Figure 5A, grey bars), and since Rapamycin induces autophagy through inhibition of mTORC1, these results indicate a function for these proteins and for miR-101 downstream of mTORC1. Further supporting this data, eGFP–LC3 translocation induced by siRNA-mediated depletion of Raptor is potently abrogated by miR-101 overexpression, additionally suggesting a function for this miRNA downstream of mTORC1 (Supplementary Figure S6). miR-101 directly targets STMN1, RAB5A and ATG4D To establish a direct molecular link, we next examined the ability of miR-101 to regulate RAB5A, STMN1 and ATG4D. As evident from Supplementary Table I, STMN1 and ATG4D hold a single 8mer seed match to miR-101 within the 3′UTR while RAB5A contains both an 8mer and a 7mer site. The predicted binding patterns of miR-101 to the 8mer motifs are depicted in Figure 6A. We cloned 300–500 base-pair 3′UTR fragments from STMN1, RAB5A and ATG4D downstream a luciferase reporter and tested the ability of miR-101 to regulate the reporters. As shown in Figure 6B, the 3′UTRs of all three putative target genes responded markedly to miR-101 overexpression relative to a scrambled control. Introducing three point mutations into the predicted miR-101 binding motifs either abolished or significantly reduced this effect, indicating that these interactions are direct. Importantly, the luciferase vectors are also s
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