Oncogene (2010) 29, 4980–4988 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc SHORT COMMUNICATION Tumor-suppressive microRNA-22 inhibits the transcription of E-box-containing c- target by silencing c-Myc binding

J Xiong, Q Du and Z Liang

Institute of Molecular Medicine, Peking University, Beijing, China

Oncogenic c-Myc has been described to modulate the Drosha and Dicer, to produce mature miRNAs. Then expression of a subset of (miRNAs), which mature miRNAs are loaded into an RNA-induced include miR-22; however, the mechanism through which a silencing complex, and associated with 30-untranslational miRNA controls c-Myc activity remains unclear. Here region (30-UTR) of mRNAs to suppress their expression we report a novel anti-c-Myc function mediated by (Bartel, 2004). The miRNAs as a set of crucial regulatory miR-22. Ectopically expressed miR-22 inhibited cell genes have diverse expression patterns, and regulate proliferation and anchorage-independent growth of human a broad spectrum of physiological and pathological cancer cell lines. Microarray screening and western processes (Esquela-Kerscher and Slack, 2006). analyses revealed that miR-22 repressed the c-Myc- Through regulating approximately 15% of all genes, binding protein MYCBP, a positive regulator of c-Myc. the Myc family of transcription factors (c-Myc, N-Myc Consistent with this, reporter assays showed that miR-22- and L-Myc) have global effects on cell proliferation and mediated MYCBP suppression largely depends growth as key oncogenic transcription factors (Cole and on the conserved miR-22 target site within the McMahon, 1999; Nesbit et al., 1999; Meyer and Penn, MYCBP 30-untranslational region (30UTR), implying 2008). The control of c-Myc expression and function that MYCBP mRNA is a direct miR-22 target. Depletion involves several mechanisms, one of which is based on of MYCBP using small interfering RNA (siRNA) the regulatory network of the c-Myc-binding . recapitulated the miR-22-induced anti-growth effect on The MYCBP (AMY-1) gene encodes a small c-Myc- tumor cells, whereas ectopically expressed MYCBP binding protein of B11 kD, that binds the N-terminal rescued cells from the growth suppression mediated by domain of c-Myc through its C-terminal region and miR-22. Moreover, repression of MYCBP by miR-22 stimulates E-box-dependent transcriptional activation downregulated a panel of E-box-containing c-Myc target of c-Myc to promote tumorigenesis (Taira et al., 1998; genes. Our results suggest that miR-22 acts as a tumor Sakamuro and Prendergast, 1999). Several miRNAs suppressor through direct repression of MYCBP expres- have been found to regulate cell proliferation and sion and subsequent reduction of oncogenic c-Myc anchorage-dependent growth (Esquela-Kerscher and activities. As c-Myc inhibits the expression of miR-22, Slack, 2006), but cross-talk between miRNAs and Myc we propose a novel positive feedback loop formed by genes has only started to emerge (Chang et al., 2008; oncogenic c-Myc to accelerate cell proliferation by Mestdagh et al., 2009). In this respect, recent studies suppressing miR-22, a potent inhibitor of MYCBP. have shown that c-Myc acts directly as a regulator of Oncogene (2010) 29, 4980–4988; doi:10.1038/onc.2010.241; a broad panel of miRNAs including miR-22 (Chang et al., published online 21 June 2010 2008; Mestdagh et al., 2009), but the effects of miRNAs on c-Myc expression and function are largely unknown. Keywords: microRNA; c-Myc binding protein; miR-22 was originally identified from HeLa cells as a MYCBP; miR-22; cancer; cell growth 22-nucleotide non-coding RNA, and was subsequently shown to be ubiquitously expressed in various tissues (Lagos-Quintana et al., 2001, 2002; Neely et al., 2006). Introduction The human miR-22 gene is located in a minimal loss of heterozygosity region between markers D17S1866 and MicroRNAs (miRNAs), a class of endogenous short (B22 D17S1574 on 17 (17p13.3) (close to TP53) nucleotides) non-coding RNAs, are highly conserved in cancer cells (Supplementary Figure 1a), and the throughout evolution (Bartel, 2004). In mammals, nascent mouse miR-22 gene is also mapped to a cancer- primary miRNAs are transcribed by RNA polymerase II associated genomic region (Suzuki et al., 2002; Calin and sequentially processed by two RNase III enzymes, et al., 2004). The human miR-22 gene overlaps the exon 2 region of the spliced non-coding C17orf91 transcript, and primary miR-22 is processed from a capped, Correspondence: Dr Z Liang, Laboratory of Nucleic Acid Technology, polyadenylated transcript (Cai et al., 2004; Rodriguez Institute of Molecular Medicine, Peking University, 5 Yiheyuan Road, et al., 2004) (Supplementary Figure 1b). In addition, the Haidian District, Beijing 100871, China. E-mail: [email protected] miRNA editing event by adenosine-deaminases acting Received 25 January 2010; revised 7 May 2010; accepted 21 May 2010; on RNA, was first described for the precursor miR-22 published online 21 June 2010 (Luciano et al., 2004) (Supplementary Figure 1c). miR-22 targeting MYCBP in cancer J Xiong et al 4981 Phylogenetic analysis showed that miR-22 is highly the two-class unpaired method in Significance Analysis of conserved evolutionarily among vertebrates, and this Microarrays (version 2.23) and filtered at a false discovery serves as an indicator for its critical biological role in rate of p0.05 (Tusher et al., 2001), and changes in the vertebrates (Supplementary Figures 1d and e). Systematic expression levels of all probes were summarized in genomic discovery of regulatory motifs in the 30-UTR of Supplementary Table 1. A previous landmark study set several mammals revealed that miR-22 ranks top among 1.4-fold as the cutoff for successful miRNA analysis all the miRNAs in terms of having the most potential (Krutzfeldt et al., 2005). Hence, mRNAs for which target genes (Xie et al., 2005). Two recent studies showed expression was downregulated more than 1.4-fold in that for a subset of human tumors, impairment of HIV- miR-22-overexpressing MCF-7 cells relative to cells transactivating response RNA-binding protein or c-Myc transfected with control RNA were selected and classified expression is correlated with defects in the expression using the categories (Supplementary Table of miRNAs including miR-22 (Chang et al.,2008;Melo 2a). Gene ontology annotations performed using Mole- et al., 2009), and this suggests a potential involvement of cular Annotation System (MAS) 2.0 software revealed miR-22 in the suppression of tumorigenesis. Therefore, we that the predominantly affected genes were enriched in the decided to examine the role of miR-22 in a tumor context. top-ranking category of ‘protein binding’ (Table 1). The TargetScan program (version 5.1) (Lewis et al., 2003) for prediction of miRNA targets was used to search Results and discussion for potential direct target genes of miR-22 from all 42 genes of the gene ontology ‘protein binding’ group. miR-22 suppresses proliferation and anchorage- Among the 19 genes that contain at least one predicted independent growth of human cancer cell lines binding site in their 30-UTR for miR-22, each of the We first determined the effect of miR-22 on human tumor genes LRRC1, DPM2, NPNT, HNRNPA3 and MYCBP cell proliferation and colony formation. A negative contains at least one putative target site that is conserved control RNA duplex with irrelevant sequences (control across mammalian species (Supplementary Table 2b). RNA, Supplementary Figure 2) was used to eliminate As miR-22 has a tumor-suppressive activity on tumor potential non-sequence-specific effects (Su et al., 2009). cell lines, and among these genes, only MYCBP was For the cell proliferation assay, MCF-7 human breast previously reported to have functions related to tumor- cancer cells were transfected with miR-22 duplex (miR-22, igenesis (Taira et al., 1998; Sakamuro and Prendergast, Supplementary Figure 2). Compared with control RNA 1999), we chose MYCBP to verify whether it is a target transfection, our results showed that miR-22 transfection of miR-22 for mediating the inhibition of proliferation induced a dramatic increase in mature miR-22 in cells and growth of tumor cell lines. MYCBP is known to (Figure 1a). It was interesting to find that cells with contribute to E-box-dependent transcriptional activation ectopically expressed miR-22 proliferated markedly more of a panel of genes by oncogenic transcription factor c- slowly than cells treated with control RNA (Figure 1b). Myc (Sakamuro and Prendergast, 1999), and thus might Subsequently, the soft agar colony assay was used to link miR-22 to its anti-growth action. MYCBP mRNA assess the capacity of miR-22 in mediating anchorage- levels were monitored in cells overexpressing miR-22 in independent cell growth, and it was shown that that comparison with cells treated with control RNA. MYCBP overexpression of miR-22 led to decreased cell numbers mRNA was substantially reduced by miR-22, which was and smaller cell colonies than control treatments well in line with the approximately twofold decrease (Figure 1c). The levels of control RNA expression observed in the Affymetrix microarray assay (Affymetrix) after colony formation were confirmed by quantitative (Figure 2a; Supplementary Figure3b).Wethenmeasured RT–PCR (Supplementary Figure 3a). the protein levels of MYCBP, and found that they were We then used MDA-MB-231 cells, a cell line that indeed lower in miR-22-overexpressing MCF-7 cells than expresses miR-22 endogenously at higher levels than in control cells in a manner that was similar to MYCBP MCF-7 cells, to evaluate the effects of anti-sense-based expression changes induced by specific small interfering miR-22 knockdown (Figure 1d). We found that the RNA (siRNA)-targeting MYCBP transcripts (Figure 2b; inhibitor of miR-22 indeed reduced the endogenous Supplementary Figure 3b). Conversely, anti-miR-22 was miR-22 levels (Figure 1e) and promoted cell growth in introduced into MDA-MB-231 cells, which express MDA-MB-231 cells (Figures 1f and g). These results miR-22 at a higher level (Figure 1c). The expression of lend support to the hypothesis that miR-22 is a potent MYCBP protein was dramatically augmented as a result inhibitor of growth in human cancer cells. of impairment of endogenous miR-22 by anti-sense compounds. This result further strengthened the hypoth- Identification of c-Myc-binding protein MYCBP esis that MYCBP is a target and an important functional as a target of miR-22 mediator of miR-22 (Figure 2c; Supplementary Figure 3c). To elucidate the mechanism of tumor suppression by miR- 22, an expression array analysis (Gene Expression Omnibus database, accession number GSE17508) was miR-22 represses MYCBP expression through direct performed on MCF-7 cells transfected with either miR-22 interaction with a conserved target site in the 30-UTR or control RNA using U133 plus 2.0 of MYCBP mRNA Array from Affymetrix (Affymetrix, Santa Clara, CA, Then we tested whether miR-22 suppresses MYCBP by USA). Differentially expressed genes were identified using direct interaction with MYCBP transcripts. Using

Oncogene miR-22 targeting MYCBP in cancer J Xiong et al 4982

Figure 1 miR-22 suppresses proliferation and anchorage-independent growth of human cancer cell lines. (a) miR-22 levels of MCF-7 cells after miR-22 transfection were measured by quantitative RT–PCR (qRT–PCR) using qRT–PCR Primer Sets (Ribobio, Guangzhou, China, catalog no. MQP-0102) with U6 small nuclear RNA (catalog no. MQP-0201), as an internal control. The PCR primers were shown to generate a single amplification band as shown in an agarose gel (Supplementary Figure 4). (b, c) Overexpression of miR-22 suppresses proliferation and anchorage-independent growth of MCF-7 cancer cells. MCF-7 cells were mock transfected, or transfected with 50 nM miR-22 or control RNA (GenePharma, Shanghai, China) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for 24 h. Then single-cell suspensions containing 10 000 cells per well were seeded in 96-well plates for bromodeoxyuridine (BrdU) proliferation assay as previously described (Lei et al., 2009) (b), and single-cell suspensions containing 10 000 cells per well were seeded in 6-well plates coated with 0.6% soft agar and maintained in Dulbecco’s modified Eagle medium (DMEM) for 2 weeks. Colonies were stained with 1.25 mg/ml nitroblue tetrazolium for 16 h, and then photographed (c, d) Expression of mature miR-22 in MCF-7 and MDA-MB-231 cancer cells measured by qRT–PCR. The normalized miR-22 expression for MCF-7 cells was set at 1. (e) miR-22 levels after anti-miR-22 transfection measured by qRT–PCR. (f, g) miR-22 knockdown promotes proliferation and anchorage-independent growth of MDA-MB-231 cells. The anti-miR-22 was a 20-O-methyl-modified oligoribonucleotide designed as an inhibitor of miR-22, and its sequence is 50-ACAGUCUUCAACUGGCAG CUU-30. The negative control for anti-miR-22 in the antagonism experiments was control anti-miR, with the sequence 50-GUGGAUAUUGUUGCCAUCA-30 (GenePharma). Transfection with 200 nM anti-miR-22 or control anti-miR was carried out for the proliferation and colony formation assay as in (b, c). BrdU proliferation assay showed a significant restoration in the presence of anti-miR-22, compared with control anti-miR (f). Upper panel: Soft agar assay showing a significant recovery in colony-forming ability of cells treated with anti-miR-22, compared with control anti-miR. Lower panel: statistical analyses (g). All data are presented as mean±standard deviation (s.d.) from at least three independent experiments. *Po0.05; **Po0.01; ***Po0.001.

TargetScan, we predicted that MYCBP has an extre- mFold-predicted DG of interaction between miR-22 mely conserved miR-22 target site (positions 38–412 nt ) and the MYCBP target site indicated that miR-22 would within its 1704 nt 30-UTR region (Figure 3a). The bind to this target with high affinity (Figure 3b). To test

Oncogene miR-22 targeting MYCBP in cancer J Xiong et al 4983 Table 1 The most affected typical gene ontology (GO) categories of potential miR-22 target genes Gene ontology ID Gene ontology description P valuea

GO:0005515 protein binding 0.0000 GO:0000166 nucleotide binding 0.0000 GO:0003924 GTPase activity 0.0000 GO:0005524 ATP binding 2.0 106 GO:0003743 Translation initiation factor activity 5.0 106 GO:0005525 GTP binding 6.0 106 GO:0046982 Protein heterodimerization activity 1.0 105 GO:0005509 Calcium ion binding 6.8 105 aP values were calculated with hypergeometric tests to determine whether there is a significant enrichment of affected genes in a GO category.

whether this site does serve as a true miR-22 target site for mediating MYCBP expression, we constructed a reporter plasmid with the whole B1.7 kb MYCBP 30-UTR (wild-type) or a version with the miR-22 target site mutated (mutant) (Figure 3c). Treatment of HEK293T or MCF-7 cells with miR-22 had a potent inhibitory effect on the luciferase activity (Figure 3d). The mRNA levels from the wild-type reporter plasmid were markedly reduced as well but this was not observed Figure 2 c-Myc-binding protein MYCBP is a target of miR-22. when the mutant reporter plasmid was used (Figure 3e). (a, b) Regulation of MYCBP expression by miR-22. MCF-7 cells were transfected with 50 nM miR-22 or MYCBP siRNAs We further measured the expression of the two reporters (MYCBP siRNA #2 sense strand 50-GUAGAAGCUAUUGUAG in MDA-MB-231 cells while manipulating endogenous AAdTdT-30, anti-sense strand 50-UUCUACAAUAGCUUCAU miR-22 levels with an anti-sense antagonist. Antagonism ACdTdT-30; MYCBP siRNA #3 sense strand 50-GGGCAU of endogenous miR-22 significantly increased the relative UAUCAGUGAACUA dTdT -30, anti-sense strand 50-UAGUUC ACUGAUAAUGCCCAA-30) (GenePharma). At 48 h post-trans- luciferase activity and mRNA levels of the wild-type fection, total RNAs were isolated using TRI Reagent (Sigma, St reporter, and this enhanced activity was completely absent Louis, MO, USA) and converted into cDNA using ImPro-II when the mutant vector was used (Figures 3f and g), Reverse Transcriptase (Promega, Madison, WI, USA) for MYCBP suggesting that this conserved target site does constitute mRNA quantification normalized against glyceraldehyde 3-phos- an essential element for MYCBP regulation by miR-22. phate dehydrogenase by qRT–PCR. Detection primers for MYCBP and GAPDH are included in Supplementary Table 3. Taken together, these functional studies suggested that Data are presented as mean±s.d. from at least three independent miR-22 directly targets the MYCBP 30-UTR to achieve its experiments (a). Total cellular proteins were prepared and silencing effect on MYCBP expression. subjected to immunoblot assay for MYCBP protein measurement relative to b-actin using antibodies for human MYCBP (Abcam, Cambridge, UK), b-actin (Santa Cruz Biotechnology, Santa Cruz, MYCBP is potentially involved in miR-22-regulated CA, USA), and an ECL kit (Santa Cruz Biotechnology). The intensity of protein bands was quantified using Image J software human cancer cell growth (National Institutes of Health, Bethesda, MD, USA) (b, c) miR-22 To investigate whether MYCBP is involved in the knockdown increases MYCBP protein yields. Transfection with regulation of human cancer cell growth by miR-22, we 200 nM anti-miR-22 or control anti-miR in MDA-MB-231 cells was designed MYCBP-specific siRNAs and constructed a carried out for the immunoblot assay as in (b). The normalized MYCBP-expressing vector containing the entire MYCBP MYCBP expression for control-RNA-treated cells was set at 1. ***Po0.001. coding sequence without the 30-UTR of MYCBP mRNA. This construct produced MYCBP protein while evading 30-UTR-mediated repressions by miR-22 (Figure 4a). First, we established that the reduction of MYCBP are mediated, at least in part, by directly limiting expression by siRNA mimicked the anti-growth effect of MYCBP expression. miR-22. The results showed that MYCBP knockdown had inhibitory effects on the proliferation and colony formation of MCF-7 cells, on a scale similar to that miR-22 might constitute a feedback loop with c-Myc found in this cell line with miR-22 overexpression. and MYCBP Moreover, overexpression of MYCBP partially relieved Previous studies have shown that MYCBP is engaged in the inhibitory effect of miR-22 on the proliferation and the activation of E-box-dependent transcription by colony formation of MCF-7 cells (Figures 4b and c; c-Myc to promote tumor cell growth (Taira et al., Supplementary Figure 3d and e). These results provide 1998; Sakamuro and Prendergast, 1999). Our study evidence that the tumor suppressive activities of miR-22 pinpointed MYCBP as a direct target of miR-22, thus

Oncogene miR-22 targeting MYCBP in cancer J Xiong et al 4984

Figure 3 miR-22 directly targets MYCBP 30-UTR. (a) MYCBP 30-UTR was predicted by TargetScan to have one conserved target site for miR-22 as well as seven other broadly-conserved miRNAs in vertebrates. (b) The miR-22 seed sequence and its target site residing at nucleotides 389–412 of the MYCBP 30-UTR. The middle seven nucleotides of miR-22 and its target region: bold; unpaired bases: above and below the duplex. (c) Diagram of MYCBP-30-UTR-containing reporter constructs. pGL3m was reconstructed from a Firefly luciferase expressing vector pGL3-control (Promega) by inserting a multiple cloning sequence immediately downstream from the Xba I site, including EcoRV,Apa I, Sac II, Nde I, Pst I, EcoR I and Nru I sites. The insertion site was immediately downstream from the stop codon of the Firefly luciferase reporter gene. A B1.7 kb fragment encoding the full-length 30-UTR of MYCBP mRNA (Genbank accession no. NM_012333) was cloned between the Xba I and Sac II sites in pGL3m, in which the putative complementary site for the miR-22 seed region was either left intact (pGL3m-MYCBP-30-UTR-WT, referred to as wild-type) or mutated ( pGL3m- MYCBP-30-UTR-Mut, referred to as Mut). Subcloning primers for MYCBP 30-UTR are included in Supplementary Table 3 and all the construct products were validated by sequencing. (d, e) Luciferase reporter assay in HEK293T and MCF-7 cells, with cotransfection of wild-type or mutant reporter, and control RNA or miR-22 as designated. At 48 h post-transfection, luciferase activity was analyzed using the Dual-Luciferase Reporter Assay System (Promega) and a Synergy HT microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT, USA), and luciferase mRNA levels were measured by qRT–PCR. The pRL-TK vector constitutively expressing Renilla luciferase was cotransfected as an internal control to minimize experimental variability caused by differences in cell viability or transfection efficiency. (f, g) Luciferase reporter assay in MDA-MB-231 cells treated with control anti-miR or anti-miR-22, was carried out as in (d, e). Transfections were done in duplicate and data are presented as mean±s.d. from at least three independent experiments. **Po0.01; ***Po0.001.

making miR-22 a likely indirect regulator of E-box- dihydroorotase, nucleolin and eukaryotic translation dependent transcription. To test this assertion, we selected initiation factor 2A. (Patel et al., 2004; Meyer and Penn, several well identified E-box-dependent target genes, 2008). These genes all lack predicted target sites of miR-22 which include cyclin D2, cyclin-dependent kinase 4, in their 30-UTR as analyzed by TargetScan, thus ruling ornithine decarboxylase, lactate dehydrogenase-A, carba- out a direct interaction between miR-22 and these genes. moyl phosphate synthase-aspartate transcarbamylase- Using an effective siRNA against MYCBP as a positive

Oncogene miR-22 targeting MYCBP in cancer J Xiong et al 4985

Figure 4 c-Myc-binding protein MYCBP is involved in miR-22-regulated human cancer cell growth. (a) Re-introduction of the MYCBP-expressing vector effectively elevated the MYCBP translational yields diminished by miR-22. The MYCBP-expressing vector (pcDNA3.1- MYCBP) was created by cloning the MYCBP coding sequence into the Xho I and EcoR I sites of pcDNA3.1 (Invitrogen). MCF-7 cells were first transfected with miR-22 for 24 h, sequentially mock transfected, or transfected with pcDNA3.1- MYCBP (indicated as MYCBP) or empty vector pcDNA3.1 (indicated as Empty vector), and then at 48 h post-transfection were subjected to immunoblot assay. Subcloning primers for MYCBP coding sequence are included in Supplementary Table 3 and the construct product was validated by sequencing. (b) miR-22 recapitulates the anti-growth effects caused by MYCBP siRNAs, and reintroduction of MYCBP partially restores the miR-22-repressed proliferation and anchorage-independent growth of human cancer cells. Transfection with control RNA or miR-22 or MYCBP siRNAs in MCF-7 cells was carried out. In rescue experiments, MCF-7 cells were initially transfected with miR-22 for 24 h and subsequently with MYCBP-expressing vector pcDNA3.1-MYCBP or empty vector pcDNA3.1. All the transfected cells were subjected to the proliferation and colony formation assay as in Figures 1b and c. BrdU assay shows a dramatic reduction of cell proliferation induced by abundant miR-22 or MYCBP siRNAs. The miR-22-mediated suppression of cell proliferation was partially rescued by reintroduction of MYCBP-expressing vector. (c) Soft agar assay indicates a dramatic reduction in colony-forming ability of cells treated with miR-22 or MYCBP siRNAs. The miR-22-mediated suppression of cell anchorage- independent growth was partially countered by reintroduction of MYCBP-expressing vector (upper panel). Statistical analyses are shown in lower panel. All data are presented as mean±s.d. from at least three independent experiments. *Po0.05; **Po0.01; ***Po0.001, compared with control RNA-transfected cells or comparison between two groups as indicated. control, we found that miR-22 had a significant inhibitory One further question we addressed was whether effect on the expression of E-box-containing genes in miR-22 interacts with c-Myc directly in the down- a way similar to siRNA-mediated knockdown of regulation of E-box-containing genes. In silico analysis MYCBP. In addition, this effect was significantly rescued using TargetScan showed no conserved binding sites for by reintroduction of an MYCBP expression vector, free miRNAs within the short c-Myc 30-UTR (467 nt), and of miR-22 repression (Figures 5a and b; Supplementary more specifically, no potential binding sites for miR-22 Figure 3f). were found through the whole length of c-Myc mRNA

Oncogene miR-22 targeting MYCBP in cancer J Xiong et al 4986

Figure 5 miR-22 might constitute a feedback mechanism with c-Myc and MYCBP. (a, b) miR-22 impairs the transcription of E-box- dependent target genes of c-Myc . Total RNAs were harvested from MCF-7 cells transfected with 50 nM control RNA or miR-22 or MYCBP siRNA for 48 h. Then mRNAs for E-box-dependent target genes of c-Myc were quantified by qRT–PCR (a) and total protein lysates were subjected to immunoblot assay (b). CCND2,cyclinD2;CDK4, cyclin-dependent kinase 4; ODC, ornithine decarboxylase; LDHA, lactate dehydrogenase-A; CAD, carbamoyl phosphate synthase-aspartate transcarbamylase- dihydroorotase; NCL, nucleolin; EIF2A, eukaryotic translation initiation factor 2A. Detection primers for the denoted target genes are included in Supplementary Table 3. (c and d)miR-22and MYCBP siRNAs had no significant impact on c-Myc expression. MCF-7 cells treated as in (a, b), were subjected to qRT–PCR for c-Myc mRNA measurement (c) and immunoblot assay for detection of endogenous c-Myc protein probed by specific anti-c-Myc antibody (Cell Signaling Technology, Beverly, MA, USA). MYCBP mRNA and protein levels were measured as positive controls. GAPDH probed by anti-GAPDH antibody (Santa Cruz Biotechnology) was used as a loading control and the relative intensity of bands was densitometrically quantified using Image J (NIH) (d). Detection primers for c-Myc mRNA are included in Supplementary Table 3. (e) The feedback regulatory model includes miR-22, c-Myc and MYCBP. (f) Phylogenetic conservation of c-Myc 30-UTR and MYCBP 30-UTR. Vista was used to generate pair wise alignments between the human genomic sequence (March 2006 assembly) and that from the indicated species. The graph is a plot of nucleotide identity for a 100-bp sliding window centered at a given position. Annotated transcripts produced from this locus are shown at the top. The 30-UTR regions are in the red box. Red indicates a conserved non-coding region; blue indicates a conserved exon; turquoise indicates conserved UTRs (upper panel). Plots representing evolutionary conservation were taken from the UCSC Genome Browser (human genome March 2006 assembly) (lower panel). Data are presented as mean±s.d. from at least three independent experiments. *Po0.05; **Po0.01; ***Po0.001, compared with control-RNA-transfected cells or comparison between two groups as indicated.

Oncogene miR-22 targeting MYCBP in cancer J Xiong et al 4987 (2379 nt). Quantitative RT–PCR and immunoblot assay by miR-22 through modulating the expression of its co- showed that c-Myc expression was not significantly factor MYCBP represents a general model for miRNA- repressed by miR-22 or MYCBP siRNAs (Figures 5c mediated regulation of c-Myc. and d). These results ruled out the possibility of a direct In summary, we report that miR-22 functions as a interaction between miR-22 and c-Myc, thus strength- tumor suppressor to strongly inhibit the growth of ening the hypothesis that miR-22 regulates E-box- human cancer cell lines, at least in part, by directly containing c-Myc target genes through direct down- targeting the 30-UTR of MYCBP mRNA. This dis- regulation of MYCBP. It is not fully established how covery allowed us to propose a loop-like regulation MYCBP regulates these E-box-containing genes because scheme that includes miR-22, c-Myc and MYCBP for of growing evidence about the function of MYCBP the control of proliferation and anchorage-independent (Furusawa et al., 2001, 2002; Yukitake et al., 2002; growth of cancer cell lines. This has expanded our Ishizaki et al., 2006), but there is evidence suggesting knowledge of the complexity of miRNA-mediated that MYCBP acts, at least in part, through modulating regulation and made it appealing to explore whether the transactivation activity of c-Myc to enhance the miRNAs fill many of the missing links within current transcription of these genes (Taira et al., 1998; gene regulatory networks. These results improve our Sakamuro and Prendergast, 1999). understanding of the functional role of miR-22 and Recent studies showed that some c-Myc-mediated suggest that it is worthwhile to explore whether miR-22 miRNAs were implicated in tumorigenesis, embryonic can serve as a therapeutic agent for the treatment of stem cell differentiation and glutamine metabolism human malignancies. (He et al., 2005; O’Donnell et al., 2005; Dews et al., 2006; Chang et al., 2008; Sander et al., 2008; Gao et al., 2009; Lin et al., 2009; Mestdagh et al., 2009). However, only two miRNAs were found to modulate c-Myc, Conflict of interest whereas c-Myc is not their significant targets (Sampson et al., 2007; Lal et al., 2009). Our results suggest a novel The authors declare no conflict of interest. schematic model for miR-22 suppression of c-Myc- mediated transactivation through targeting MYCBP in a feedback manner (Figure 5e). Comparative genomics assay showed that all c-Myc genes from higher Acknowledgements eukaryotic species have evolved to bear only an extre- mely short 30-UTR, which could presumably serve as We thank Tianjing Cai for excellent technical assistance, and a perfect strategy for a gene to avoid direct post- Dr Iain C Bruce for critical reading of the paper. This work was supported by the National High-tech R&D Program of transcriptional regulation by the miRNA repertoire. In 0 China (2007AA02Z165, 2008DFA30770), the National Basic contrast, MYCBP retains a relatively lengthy 3 -UTR Research Program of China (2007CB512100), the National (1704 nt) that could accommodate more miRNA ma- Natural Science Foundation of China (30873187, 30771085 nipulation (Figure 5f). These findings prompted the and 30871385) and by grants from the Department of hypothesis that the indirect regulation of c-Myc activity Education of China (20070001011 and 200800010019).

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