A component of the mir-17-92 polycistronic oncomir promotes oncogene-dependent apoptosis

mir-17-92, a potent polycistronic oncomir, encodes six mature miRNAs with complex modes of interactions. In the Eμ-myc Burkitt’s lymphoma model, mir-17-92 exhibits potent oncogenic activity by repressing c-Myc-induced apoptosis, primarily through its miR-19 components. Surprisingly, mir-17-92 also encodes the miR-92 component that negatively regulates its oncogenic cooperation with c-Myc. This miR-92 effect is, at least in part, mediated by its direct repression of Fbw7, which promotes the proteosomal degradation of c-Myc. Thus, overexpressing miR-92 leads to aberrant c-Myc increase, imposing a strong coupling between excessive proliferation and p53-dependent apoptosis. Interestingly, miR-92 antagonizes the oncogenic miR-19 miRNAs; and such functional interaction coordinates proliferation and apoptosis during c-Myc-induced oncogenesis. This miR-19:miR-92 antagonism is disrupted in B-lymphoma cells that favor a greater increase of miR-19 over miR-92. Altogether, we suggest a new paradigm whereby the unique gene structure of a polycistronic oncomir confers an intricate balance between oncogene and tumor suppressor crosstalk. DOI: http://dx.doi.org/10.7554/eLife.00822.001


Introduction
MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate post-transcriptional gene repression in a variety of developmental and pathological processes (Ambros, 2004;Zamore and Haley, 2005;Bartel, 2009;Kim et al., 2009). Due to their small size and the imperfect nature of target recognition, miRNAs have the capacity to regulate many target mRNAs through translational repression and mRNA degradation, thereby acting as global regulators of gene expression (Lewis et al., 2005;Filipowicz et al., 2008). Unlike mammalian protein-coding genes that follow the one-transcript, one-protein paradigm, many miRNA genes are expressed as polycistronic primary transcripts, generating multiple mature miRNAs under the same transcriptional regulation (Megraw et al., 2007). miRNA polycistrons further expand the gene regulatory capacity, since different miRNA components can confer specific yet overlapping biological effects, and their functional interactions can yield unusual complexity.
The structural analogy to prokaryotic operons has led to the speculation that the co-transcribed mir-17-92 components can collectively contribute to oncogenesis. However, our studies reveal an unexpected functional interaction among mir-17-92 components. In the Eμ-myc mouse B-cell lymphoma model, while the intact mir-17-92 acts as an oncogene, its miR-92 component negatively regulates the oncogenic cooperation with c-Myc. This effect, at least in part, results from the ability of miR-92 to eLife digest The role of genes, in very simple terms, is to be transcribed into messenger RNA molecules, which are then translated into strings of amino acids that fold into proteins. Each of these steps is extremely complex, and a wide range of other molecules can speed up, slow down, stop or otherwise disrupt the expression of genes as protein products. Genes can also code for nucleic acids that are not translated into proteins, such as microRNAs. These are small RNA molecules that can reduce the production of proteins by repressing the translation step and/or by partially degrading the messenger RNA molecules.
mir-17-92 is a gene that exemplifies much of this complexity. It codes for six different microRNAs in a single primary transcript, and has been implicated in a number of cancers, including lung cancer, Burkitt's lymphoma and other forms of lymphomas and leukemia. One of six microRNAs has a longer evolutionary history than the remaining five: mir-92 is found in vertebrates, chordates and invertebrates, whereas the other five are only found in vertebrates. However, it is not known how or why the mir-17-92 gene evolved to code for multiple different microRNAs.
Olive et al. have studied how these mir-17-92 microRNAs functionally interact in mice with Burkitt's lymphoma, a form of cancer that is associated with a gene called c-Myc being over-activated. Mutations in this gene promote the proliferation of cells, and in cooperation with other genetic lesions, this ultimately leads to cancer. mir-17-92 is implicated in this cancer because it represses the process of programmed cell death (which is induced by the protein c-Myc) that the body employs to stop tumors growing. Olive et al. found that deleting one of the six microRNAs, miR-92, increased the tendency of the mir-17-92 gene to promote Burkitt's lymphoma. By repressing an enzyme called Fbw7, miR-92 causes high levels of c-Myc to be produced. While this leads to the uncontrolled proliferation of cells that promotes cancer, it also increases programmed cell death, at least in part, by activating the p53 pathway, a well-known tumor suppression pathway. The experiments also revealed that the action of miR-92 and that of one of the other microRNAs, miR-19, were often opposed to each other. These findings have revealed an unexpected interaction among different components within a single microRNA gene, which acts to maintain an intricate balance between pathways that promote and suppress cancer.  polycistron and its mutated derivatives. Light colored boxes, pre-miRNAs; dark colored boxes, mature miRNAs. Homologous miRNA components are indicated by the same color. (B) Schematic representation of the adoptive transfer protocol using Eμ-myc hematopoietic stem and progenitor cells (HSPCs). Eμ-myc/+ HSPCs were extracted from E13.5-E15.5 mouse embryos, infected with MSCV retroviral vectors overexpressing mir-17-92 and its derivatives, and finally transplanted into lethally irradiated recipient mice. Lymphoma onset of the adoptive transferred mice was monitored to evaluate the oncogenic collaboration between c-Myc and a specific miRNA. (C) miR-92 deficiency specifically accelerates the oncogenic activity of mir-17-92 in the Eμ-myc model. Using the Eμ-myc adoptive transfer model, we compared the oncogenic effects between mir-17-92 and mir-17-92Δ92 and observed a significant acceleration of tumor onset in left). When the oncogenic effects of mir-17-92, Figure 1. Continued on next page yield aberrant c-Myc dosage, which promotes a strong coupling between oncogene stress and p53-dependent apoptosis. Surprisingly, miR-92 functionally antagonizes miR-19, a key oncogenic mir-17-92 component, in the context of c-Myc-induced oncogenesis. During B-cell transformation, this miR-19:miR-92 antagonism is disrupted to favor a greater increase of miR-19 than miR-92. Thus, the polycistronic mir-17-92 employs an antagonistic interaction among its encoded miRNA components to confer an intricate crosstalk between the oncogene and tumor suppressor networks.
During Myc-induced tumorigenesis, aberrant c-Myc dosage yields simultaneous induction of proliferation and apoptosis, imposing a unique selective pressure for pro-survival lesions (Evan and Vousden, 2001). Thus, we compared the extent of Myc-induced apoptosis in the , and control Eμ-myc/MSCV lymphomas. The control Eμ-myc/MSCV lymphomas invariably exhibited a high proliferation index accompanied by extensive cell death, as evidenced by the widespread 'starry sky' pathology ( Figure 2C,D) and cleaved caspase 3 staining ( Figure 2C,E). The potent oncogenic activity of mir-17-92Δ92 and mir-17-92Mut92 was consistent with the strong reduction of apoptosis in the lymph node tumors. In comparison, the intact miR-92 significantly attenuated the repression of c-Myc-induced apoptosis by mir-17-92 in vivo ( Figure 2C-E).
We next investigated the effect of miR-92 alone in regulating c-Myc-induced apoptosis. In the Eμ-myc model, miR-92 overexpression significantly enhanced c-Myc-induced apoptosis in vivo ( Figure 3A (Murphy et al., 2008). The R26 MER/MER MEFs recapitulate c-Myc-induced apoptosis in vitro, as activated MycER T2 induces p53-dependent apoptosis in response to serum starvation (Murphy et al., 2008). In addition to promoting c-Myc-induced apoptosis, miR-92 unexpectedly enhanced c-Myc-induced cell proliferation. A significant increase of BrdU incorporation was observed in R26 MER/MER MEFs overexpressing miR-92, both under normal culture conditions and, more evidently, under serum starvation ( Figure 3D). The same proliferative effect of miR-92 was also observed in primary B-cells. Comparison of the proliferative effect of each mir-17-92 component in bone marrow derived primary dissemination, as indicated by H&E and B220 staining. (C) Eμ-myc/17-92Mut92 and Eμ-myc/17-92Δ92 lymphomas exhibited a decreased apoptosis compared to Eμ-myc/MSCV or Eμ-myc/17-92 lymphomas. Representative lymphomas were stained for H&E, cleaved caspase-3 and PCNA. Arrow, 'starry sky' feature of apoptotic lymphoma cells; arrowhead, apoptotic cells with positive staining for cleaved caspase-3; scale bar, 50 μm. (D and E) Apoptosis was quantitatively measured in representative lymphomas of each genotype using the 'starry sky' features (D) and cleaved caspase-3 staining (E). *p<0.05, **p<0.01, ***p<0.001. DOI: 10.7554/eLife.00822.005   To investigate the molecular mechanism underlying miR-92 functions, we performed microarray analyses comparing gene expression profiles of R26 MER/MER MEFs overexpressing miR-92 or the control MSCV vector. These MEFs were serum starved and 4-OHT treated to trigger strong Myc-induced apoptosis. miR-92-upregulated genes were significantly enriched for the cell cycle pathway, including ccnd1, ccnb1, ccnb2, cdc25b, cdc25c, and cdk4 ( Figure 4A,B), consistent with the ability of miR-92 to promote Myc-induced cell proliferation. Genes upregulated by miR-92 were also enriched for the p53 pathway, including the classic p53 target mdm2, as well as the pro-apoptotic p53 targets-noxa, bax, puma, perp, and bid ( Figure 4A,B, Figure 4-figure supplement 1A). Since aberrant c-Myc activation triggered a p53-dependent apoptotic response (Lowe et al., 2004), our observation is consistent with miR-92 further enhancing p53 activation downstream of c-Myc. Interestingly, p21, a canonical p53 target, was not induced by miR-92 in the MycER T2 activated R26 MER/MER MEFs (Figure 4-figure supplement 1A). It is likely that the transcriptional repression of p21 by c-Myc renders p21 irresponsive to p53 activation under this biological context (Heasley et al., 2002). Using real-time PCR, we validated the ability of miR-92 to induce cell cycle genes and activate p53 targets in both R26 MER/MER MEFs, as well as primary B-cells ( Figure 4C, Figure 4-figure supplement 1A,B). Hence, the molecular signature imposed by miR-92 overexpression is consistent with its functional readout.
The activation of the p53 pathway by c-Myc is essential for the induction of the apoptotic response in the Eμ-myc model (Schmitt et al., 2002). A major mechanism that governs Myc-induced p53 activation is the transcriptional induction of the gene encoding Arf, which inhibits Mdm2mediated p53 ubiquitination and degradation (Lowe et al., 2004;Campaner and Amati, 2012). The ability of miR-92 to enhance c-Myc-induced apoptosis and to increase the expression of p53 targets raised the possibility that miR-92 overexpression activates p53 possibly through elevated Arf. In both R26 MER/MER MEFs and wild-type primary B-cells, miR-92 overexpression alone caused significant accumulation of Arf mRNA and protein ( Figure  reconstituted from donor matched Eμ-myc HSPCs. Premalignant Eμ-myc splenic B-cells were isolated from the Eμ-myc/MSCV and Eμ-myc/92 mice 6 weeks after reconstitution. The in vivo apoptosis was measured by the level of caspase activation using Red-VAD-FMK, a fluorescently labeled caspase inhibitor that specifically bound to cleaved caspases. The percentage of Eμ-myc B-cells positive for cleaved caspases was shown for four independent experiments. (C) Enforced miR-92 expression in R26 MER/MER MEFs significantly enhanced c-Myc-induced apoptosis. miR-92 overexpressing and the control R26 MER/MER MEFs were serum starved, and the MycER T2 transgene was activated by 4-OHT treatment. The level of apoptosis of each MEF was measured using Annexin V staining before (left) and after (middle) 4-OHT treatment and serum starvation. Quantification of c-Myc-induced apoptosis was performed in three independent MEF lines that overexpressed MSCV or miR-92 (   The induction of p53 by miR-92 prompted us to investigate the functional importance of p53 in miR-92-induced apoptotic response. Knockdown of p53 in R26 MER/MER MEFs not only led to a suppression of c-Myc-induced apoptosis, but also completely abolished the effect of miR-92 to enhance c-Mycinduced apoptosis ( Figure 4E). These findings suggested that an intact p53 pathway is required for the apoptotic effect of miR-92. Consistently, the miR-92 induction of the pro-apoptotic genes, including noxa, perp, and mdm2, also was mediated by the intact p53 ( Figure 4F). Thus, aberrant c-Myc activation triggers an apoptotic response through p53 activation; and co-expression of miR-92 with c-Myc leads to an even stronger p53 activation, and subsequently apoptotic response.
Our findings suggest parallels between c-myc and miR-92: both are potent oncogenes that promote excessive cell proliferation coupled with p53-dependent apoptosis, and both are capable to induce expression of cell cycle genes (ccnb1, ccnd1, cdk4, and cdc25) (Lowe et al., 2004;Campaner and Amati, 2012) and p53 pathway components (Arf, puma, noxa, perp, and mdm2) (Lowe et al., 2004;Campaner and Amati, 2012). The functional analogy between c-Myc and miR-92, as well as the molecular overlap between their downstream pathways, led us to investigate the effect of miR-92 on c-Myc. Intriguingly, miR-92 expression significantly enhanced c-Myc protein level both in MEFs and in primary B-cells ( Figure 5A), without affecting the c-myc mRNA level ( Figure 5-figure supplement 1A, data not shown). Consistent with the stabilization of endogenous c-Myc, miR-92 overexpression in R26 MER/MER MEFs stabilized the MycER T2 protein ( Figure 5B). The dosage of c-Myc protein is crucial for its biological readout (Murphy et al., 2008). While c-Myc dosage determines the extent of cell cycle gene induction and cell proliferation, it also regulates the degree of p53 activation and subsequent apoptosis (Murphy et al., 2008) (Figure 5-figure supplement 1B). Thus, the ability of miR-92 to induce aberrant c-Myc accumulation likely constitutes the molecular basis for its ability to promote both cell proliferation and p53-dependent apoptosis.
Based on our findings, we speculated that miR-92 targets could include negative regulators of c-Myc protein accumulation. Therefore, we searched genes known to negatively regulate c-myc for the presence of putative miR-92 binding sites. Using the Targetscan and RNA22 algorithms (Lewis et al., 2005;Miranda et al., 2006;Bartel, 2009), we identified eight candidate miR-92 targets, each of which contained one or more predicted miR-92 binding sites in the 3′ untranslated region (3′UTR). Real-time PCR analysis of these candidate genes confirmed fbw7 (F-box and WD repeat domaincontaining 7) as a likely target of miR-92 ( Figure 5C, Figure 5-figure supplement 1C). fbw7, which contains two miR-92 target sites within its 3′UTR ( Figure 5C), is the substrate recognition component of an SCF-type E3 ubiquitin ligase that mediates the degradation of several proto-oncoproteins, including Myc, Cyclin E, c-Jun, and Notch (Welcker and Clurman, 2008;Crusio et al., 2010;Wang et al., 2012). A luciferase reporter or a FLAG-tagged fbw7-encoding ORF (open reading frame), when fused to the wild-type fbw7 3′ UTR, were both significantly repressed in a miR-92 dependent manner ( Figure 5D, Figure 5-figure supplement 1D). Yet enforced miR-92 expression failed to repress the luciferase reporter that contained an fbw7 3′UTR with two mutated miR-92 binding sites ( Figure 5D), suggesting that miR-92 binding to fbw7 3′UTR is required for this repression. Furthermore, miR-92 effectively repressed endogenous Fbw7 protein level, as demonstrated by the decreased fbw7 mRNA level and  Fbw7 immunoprecipitation ( Figure 5E). Consistent with fbw7 as an important target for miR-92, enforced miR-92 expression upregulated multiple Fbw7 substrates at their protein levels, including c-Myc and cyclinE ( Figure 5F). Observations from our in vivo experiments also supported this post-transcriptional regulation of Fbw7 by miR-92, as we observed an inverse correlation between the level of miR-92 and fbw7 when comparing Eμ-myc/17-92Δ92 and Eμ-myc/17-92 lymphoma cells ( Figure  5-figure supplement 1E).
fbw7 has previously been postulated as a potential miR-92 target based on the presence of miR-92 target sites (Mavrakis et al., 2011), yet it remains unclear how fbw7 mediated the pro-apoptotic effects of miR-92, given its well-characterized functions as a tumor suppressor. Recent findings indicate that the acute inactivation of tumor suppressor Fbw7 imposes a strong oncogenic stress to induce p53-dependent apoptosis, conferring a selective advantage to cells with deficient p53 function (Minella et al., 2007;Onoyama et al., 2007;Matsuoka et al., 2008;Grim et al., 2012). This p53-dependent apoptosis is, at least in part, due to an aberrant increase of c-Myc dosage (Onoyama et al., 2007;Matsuoka et al., 2008). These findings suggested that a major mechanism through which miR-92 enhanced the c-Myc protein level, and subsequently, c-Myc-induced apoptosis, could be through its direct repression of Fbw7. In support of this hypothesis, miR-92 overexpression significantly increased the c-Myc protein level in wild-type Hct116 cells, but not in FBW7 −/− Hct116 cells ( Figure 5-figure  supplement 1F), suggesting FBW7 was essential for miR-92 to induce c-MYC increase. Functionally, acute fbw7 knockdown in R26 MER/MER MEFs partially phenocopied the effect of miR-92 to enhance c-Myc-induced apoptosis ( Figure 5G, Figure 5-figure supplement 1G); while overexpression of an fbw7α open reading frame (ORF), albeit above its physiological level, completely abolished this apoptotic effect of miR-92 ( Figure 5H, Figure 5-figure supplement 1H). Nevertheless, it is still likely that additional mechanisms downstream of miR-92 also promote its apoptotic effects, because fbw7 knockdown largely recapitulated the extent of c-Myc upregulation by miR-92 ( Figure 5-figure  supplement 1G), yet only partially phenocopied its pro-apoptotic effects ( Figure 5G). In addition, overexpression of fbw7 above its physiological level might amplify the extent of functional interactions between fbw7 and miR-92 in regulating apoptosis. Despite these caveats, our results strongly argue that the miR-92-Fbw7 axis constitutes a major mechanism underlying the pro-apoptotic effects of miR-92.
Downregulation of Fbw7 by miR-92 significantly enhanced the protein level of c-Myc in R26 MER/MER MEFs and in primary B-cells ( Figure 5A). It is conceivable that the ability of miR-92 to repress Fbw7 in vivo could similarly enhance the c-Myc accumulation in the Eμ-myc/92 premalignant B-cells, promoting rapid cell proliferation and a p53-dependent apoptotic response. Unfortunately, due to technical limitations, approach in R26 MER/MER MEFs has provided us with the most consistent measurement for c-Myc protein level, because it is regulated in a cell-cycle-dependent manner. (B) miR-92 overexpression decreases the turnover of c-Myc protein. Serum-synchronized R26 MER/MER MEFs that overexpress either miR-92 or the control MSCV vector were released into the serum for 6 hr, treated with cycloheximide, collected at the indicated time points, then analyzed by western blot to determine the levels of MycER and the endogenous c-Myc protein. (C) Schematic representation of the two miR-92 binding sites in the murine fbw7 3′UTR. Additionally, a luciferase reporter and a FLAG tagged fbw7 ORF were each placed upstream of a wild-type fbw7 3′UTR, or a mutated fbw7 3′UTR that abolished the predicted miR-92 binding. (D) The expression of Luc-fbw7-3′UTR was specifically repressed by miR-92 in Dicer −/− HCT116, while mutations of the two putative miR-92 binding sites within the fbw7-3′UTR (Luc-fbw7-3′UTRMut)   we were not able to demonstrate an increased c-Myc protein level as a result of miR-92 overexpression in the Eμ-myc premalignant B-cells. There was a significant depletion of the Eμ-myc/92 premalignant B-cells due to excessive apoptosis (Figure 3-figure supplement 1B), making it difficult to collect enough cells to analyze the c-Myc protein level by western analyses. Similarly, we could not obtain enough cells to compare the protein level of c-Myc in the premalignant B-cells from the Eμ-myc/17-92, Eμ-myc/17-92Δ92, and Eu-myc/MSCV animals. Nevertheless, our functional studies in cell culture, combined with the inverse expression correlation between fbw7 and miR-92 in vivo, strongly argue the importance of the miR-92-Fbw7-Myc axis to promote the pro-apoptotic effects of miR-92.
In the context of the c-Myc cooperation, mir-17-92 encodes miRNA components with opposing biological functions. While miR-19 miRNAs repress c-Myc-induced apoptosis to promote Eμ-myc lymphomagenesis (Mu et al., 2009;Olive et al., 2009), miR-92 enhances c-Myc-induced apoptosis to attenuate the tumorigenic effects. Consistent with the opposing effects of miR-19 and miR-92, co-expression of these two miRNAs as a dicistron attenuated the apoptotic effect of miR-92 in premalignant Eμ-myc B-cells in vivo ( Figure 6A,B, Figure 6-figure supplement 1A). A similar antagonistic interaction was also observed in R26 MER/MER MEFs; and introducing a miR-19b mutation within mir-19b-92 dicistron abolished this interaction ( Figure 6C). Since miR-19 represses pten to promote the PI3K/AKT pathway, the activation of AKT signaling would lead to increased phosphorylation of Mdm2, thus destabilizing p53 to dampen the apoptotic response induced by miR-92 (Gottlieb et al., 2002;Ogawara et al., 2002). Consistent with this hypothesis, we observed a decreased p53 induction and an unaltered c-Myc level when miR-92 was co-expressed with miR-19 ( Figure 6D, Figure 6figure supplement 1B).
This miR-19:miR-92 antagonism appears to be conserved evolutionarily. In Xenopus laevis, miR-19 and miR-92 have identical sequence to their mammalian orthologs (Figure 1-figure supplement 1B). Based on Targetscan and RNA22 miRNA target prediction algorisms (Lewis et al., 2003(Lewis et al., , 2005Miranda et al., 2006;Grimson et al., 2007), their target specificity is also conserved for key miRNA targets, although the exact binding sites may or may not be conserved (Olive et al., 2009) (Figure 6figure supplement 1C). In addition, the biological functions of miR-19 and miR-92 exhibit evolutionary conservation between Xenopus laevis and mammals. Individual injection of miR-19 promoted cell survival of hydroxyurea-treated Xenopus embryos, while co-injection of miR-19a and miR-92 significantly attenuated this pro-survival effect ( Figure 6E). This functional antagonism was specific for miR-92 and miR-19, since co-injection of other mir-17-92 components or a mutated miR-92 did not yield any functional interactions in combination with miR-19 ( Figure 6F).
Given the opposing biological effects of miR-19 and miR-92 during c-Myc-induced lymphoma development, differential regulation of these two miRNA families could determine the oncogenic activity of mir-17-92. Under normal physiological conditions, this miR-19:miR-92 antagonism could attenuate the detrimental oncogenic signaling by inducing apoptosis in cells with inappropriate mir-17-92 induction. During malignant transformation, and particularly during c-Myc-induced oncogenesis, this miR-19:miR-92 antagonism could be disrupted to favor cell survival. Using real time PCR analyses, we compared the relative abundance of miR-19a, miR-19b, and miR-92 in normal splenic B-cells, premalignant Eμ-myc B-cells, and Eμ-myc lymphomas ( Figure 7A). Comparing to normal splenic B-cells, the levels of all three mature miRNA species were elevated in both premalignant and malignant Eμ-myc B-cells, possibly due to transcriptional activation of mir-17-92 by c-Myc (Donnell et al., 2005). However, the miR-19 to miR-92 ratios significantly increased during c-Myc-induced lymphomagenesis ( Figure 7B). In other words, when normalized to the respective miRNA levels in normal splenic B-cells, mature miR-19 (including miR-19a and miR-19b) exhibited a greater increase in premalignant and malignant Eμ-myc B-cells than mature miR-92 ( Figure 7A-C). This differential increase was most evident in premalignant Eμ-myc B-cells; the fully transformed Eμ-myc B-lymphoma cells exhibited a lesser difference ( Figure 7A,B). This observation is consistent with premalignant Eμ-myc B-cells having an intact p53-dependent apoptotic response, thus a stronger selective pressure for a greater miR-19:miR-92 ratio. In comparison, most Eμ-myc B-lymphomas have a defective p53 response, hence a less strong selective pressure to maintain a high miR-19:miR-92 ratio. We also validated this observation using northern analysis. Comparing normal splenic B-cells and multiple Eμ-myc lymphoma cells, the levels of the mature miR-19a, miR-19b and miR-92 were all elevated in transformed B-cells; however, the degree of increase for miR-19a and miR-19b was significantly higher than that of miR-92 ( Figure 7C). This differential increase of miR-19 and miR-92 was also observed in human Burkitt's lymphoma cell lines when compared to normal B-cells isolated from the periphery blood ( Figure 7D). More importantly, this phenomenon was not limited to  Figure 7E).
These observations were consistent with a previous finding, where the inducible c-myc activation in a human Burkitt's lymphoma cell line induced both miR-19a and miR-19b to a greater extent than miR-92 (Donnell et al., 2005). Although miR-19 and miR-92 are co-transcribed from the mir-17-92 precursor, the differential increase of miR-19 vs miR-92 occurs in multiple c-Myc-driven tumor types. Thus, the relative abundance of miR-19 and miR-92 could constitute an important molecular basis to regulate the initiation and progression of c-Myc-induced tumor development.

Discussion
The unique polycistronic structure of mir-17-92 constitutes the basis for its pleiotropic functions and the complex mode of interactions among its miRNA components. A high level of mir-17-92 in normal or premalignant cells could lead to suboptimal consequences that are counter-balanced through an intrinsic negative regulation by miR-92 ( Figure 7F). As we demonstrated in vitro where miR-92, by directly downregulating Fbw7, enhances c-Myc protein level to promote apoptosis, the ability of miR-92 to repress Fbw7 in vivo could similarly constitute a major mechanism to enhance c-Mycinduced apoptosis. This effect of miR-92 is a double edged sword in c-Myc driven tumors, as its overexpression gives rise to a strong and obligated coupling between excessive proliferation and a potent, p53-dependent apoptosis ( Figure 7F). This coupling is consistent with the previous observation that a lower level of constitutive c-Myc acts more effectively to promote tumor initiation, while a higher level of c-Myc is selected by the terminal tumors with defective apoptosis machinery (Murphy et al., 2008). Therefore, mir-17-92 encodes an internal component to confer a negative regulatory feedback on its oncogenic activity, imposing a strong selection for anti-apoptotic lesions to shape the path of malignant transformation. More interestingly, c-Myc transcriptionally activates mir-17-92 that encodes miR-92 (Hemann et al., 2005), which in turn enhances c-Myc dosage, at least in part, by repression Fbw7. It is possible that aberrant c-Myc activation triggers a positive feedback loop to further increase c-Myc dosage to strengthen the apoptotic response and to eliminate cells with oncogenic potential. It is worth noting that the miR-92 apoptotic effect described in this study depends on an intact p53 response. Consequently, in terminal Eμ-myc B-lymphoma cells that often carry a defective p53 response, miR-92 failed to enhance c-Myc-induced apoptosis (Mu et al., 2009).
The functional readout of miR-92 heavily depends on cell types and biological contexts. It is important to recognize that miR-92 is not a tumor suppressor miRNA. Like c-Myc, miR-92 elicits potent oncogene stress to engage tumor suppressor response, at least in part, by activating p53. In the premalignant Eμ-myc/92 B-cells, the effect of miR-92 to repress Fbw7 most likely results in an increase of c-Myc level, which coupled with the intact p53 response to strongly sensitize the cells to miR-92-induced apoptosis.

adoptive transfer model enhanced apoptosis in premalignant Eμ-myc splenic B-cells, while the mir-19b-92 dicistron expression abolished this apoptotic effect (left three panels). A quantitative analysis of apoptosis by FACS was shown for three independent, well-controlled experiments (right). (C) miR-19b
dampens the miR-92-induced apoptosis in MycER T2 activated R26 MER/MER MEFs. R26 MER/MER MEFs were infected by  and the MSCV control vector, and were subsequently serum starved and treated with 4-OHT to activate MycER T2 . Apoptosis in these samples was measured quantitatively using Annexin V staining (left four panels). The extent of apoptosis induced by MSCV, was normalized to that of MSCV infected R26 MER/MER MEFs and then averaged from four independent experiments (right). (D) miR-19b dampens the miR-92-induced p53 activation. R26 MER/MER MEFs that overexpress the indicated constructs  were collected 48 hr after infection and then analyzed by western blot to determine the level of p53 protein. (E) miR-92 and miR-19 exhibit antagonistic effects to regulate hydroxyurea (HU)-induced cell death in Xenopus embryos. Representative images of HU-treated Xenopus embryos that were co-injected with human Ago2 and the indicated miRNA mimics (left). Co-injection of miR-92 dampened the cell survival effects of miR-19 on HU-induced apoptosis (right, n = 3, with >20 embryos in each group). (F) miR-92 exhibits a specific antagonistic interaction with miR-19. Injection of miR-19a or miR-19b rescued HU-induced apoptosis in Xenopus embryos. Co-injection of miR-92, but not a mutated miR-92, or other mir-17-92 components, dampened the cell survival effect of miR-19 (n = 3, with >20 embryos in each group). Error bars represent standard deviation, *p<0.05; **p<0.01. DOI: 10.7554/eLife.00822.013 The following figure supplements are available for figure 6:  Under other contexts when proliferation becomes a rate-limiting event for oncogenesis, or when p53-dependent apoptosis is compromised, miR-92 could render a pro-proliferative effect that is strictly oncogenic (Tsuchida et al., 2011). Likewise, the functional readout of other mir-17-92 components also heavily depends on cell types and biological contexts. miR-19 promotes c-Myc-induced B-lymphomas by repressing apoptosis (Mu et al., 2009;Olive et al., 2009), yet has little effects in promoting Rb-deficient retinoblastomas (Conkrite et al., 2011); miR-17 allows the bypass of Rasinduced senescence by promoting proliferation (Hong et al., 2010), yet fails to affect c-Myc-induced lymphomas, possibly due to its functional redundancy with c-Myc.
Both cooperative and antagonistic interactions operate among subsets of mir-17-92 components. The miR-19:miR-92 antagonism constitutes a novel mechanism to confer an intricate balance between oncogene signaling and innate tumor suppressor responses ( Figure 7F). This balance can be disrupted in premalignant and malignant cells that exhibit c-Myc overexpression, as an increase in the miR-19:miR-92 ratio is likely to favor the suppression of c-Myc-induced apoptosis and to promote oncogenesis. Although all mir-17-92 components are co-transcriptionally regulated, different changes of miR-19 vs miR-92 during oncogenesis could be a result of differential miRNA biogenesis and/or turn-over. It has been shown that specific RNA-binding proteins, such as hnRNP A1, promote the processing of a specific mir-17-92 component, miR-18 (Guil and Cáceres, 2007). Future studies are likely to reveal important mechanisms underlying cell type-and context-dependent differential regulation of mir-17-92 components, which will generate important insights on the biology of polycistronic miRNAs.
Our current study mostly focuses on the antagonistic interaction between miR-19 and miR-92 in c-Myc driven oncogenesis, yet it reveals a more general mechanism underlying the structural function relationship of polycistronic miRNAs. It is likely that the complex interactions among polycistronic miRNA components can coordinate and balance a multitude of cellular and molecular processes during normal development and disease. Interestingly, in the case of mir-17-92, miR-92 has a different evolutionary history compared to the other mir-17-92 components. miR-92 is evolutionary conserved in Deuterostome (including vertebrates and chordates), Ecdysozoa (including flies and worms), and Lophotrochozoa, yet the remaining mir-17-92 components are only found in vertebrates (Figure 1figure supplement 1C). The functional antagonism between the more ancient miR-92 and the newly evolved mir-19 might result from the convergence of these two separate evolutionary paths at the origin of vertebrates. This antagonism could evolve to regulate cell proliferation and cell death downstream or independent of c-Myc in both normal development and disease. Thus, our studies suggest a novel mechanism by which a crosstalk between oncogene and tumor suppressor pathways has been hardwired through evolution into the unique gene structure of a polycistronic oncomir.

Molecular cloning
mir-17-92 Δ92 and mir-17-92 were amplified by PCR and subsequently cloned into the XhoI and EcoRI sites of the MSCV retrovirus vectors. In these vectors, miRNAs were placed downstream of the LTR miR-19a, miR-19b and miR-92. Comparing premalignant/malignant Eμ-myc B-cells vs normal splenic B-cells, all three miRNAs exhibited an increased level, although the increase in miR-19a or miR-19b was significantly higher than that of miR-92 (A). In the same experiment, the relative ratios for miR-19a:miR-92 and miR-19b:miR-92 were measured for all normal splenic B
The loss of miR-92, miR-20 or miR-19b expression and the intact expression level of the remaining mir-17-92 components were validated using the TaqMan MicroRNA Assays (4427975; Applied Biosystems, Foster City, CA).  were similarly amplified by PCR (ACTGCTCGAGAGCTTCGGCCTGTCGCCC and GTAGAATTCATGTATCTTGTAC) from the mir- , and mir-17-92Mut19b construct described above and subsequently cloned into the XhoI and EcoRI sites of the MSCV retrovirus vectors.

Adoptive transfer of Eμ-myc HSPCs for lymphomagenesis
The hematopoietic stem and progenitor cells (HSPCs) were isolated from E13.5-E15.5 Eμ-myc/+ mouse embryos and were transduced with MSCV alone or MSCV vectors expressing various mir-17-92 derivatives. The MSCV retroviral vector used in our adoptive transfer model contains a SV40-GFP cassette that allows us to monitor transduced HSPCs both in vitro and in vivo. Infected HSPCs were subsequently transplanted into an 8-to 10-week-old, lethally irradiated C57BL/6 recipient mice. Tumor onset was subsequently monitored by weekly palpation, and tumor samples were either collected into formalin for histopathological studies, or prepared as single cell suspension for FACS analysis and for cell culture studies. Both the Eμ-myc/+ mice and the recipient mice are on C57BL/6 background.

LT2-MYC mouse liver tumor model
The LT2-MYC mouse model for human hepatocellular carcinoma (HCC) is a double transgenic mouse model, in which the tetracycline transactivator protein (tTA) is driven by the hepatocyte-specific promoter, the liver activator protein (LAP) promoter, while the human c-MYC gene is driven by the tetracycline response element (TRE). The LT2-MYC model exhibits 'dox-off' regulation, where c-Myc expression is turned on in hepatocytes in the absence of doxycycline.
LT2-MYC mice taken off doxycycline-containing food, between 3-5 weeks of age, develop distinct tumor nodules around 8-12 weeks on an average (Kistner et al., 1996;Shachaf et al., 2004). Total RNA was extracted from liver tumor samples from three independent mice, as well as normal livers from the doxycycline treated LT2 mice. Total RNAs were prepared using Trizol (15596018; Invitrogen) and subjected to real time PCR analyses as described below.
Mouse primary B-cell cultures or MEFs were infected by MSCV retroviruses expressing various mir-17-92 derived miRNA clusters, shRNA against p53 (Xue et al., 2007), shRNA against fbw7 (Mavrakis et al., 2011), or fbw7 cDNA (pRetroX-fbw7-IRES-DsRedExpress). In Figure 4E,F, double infection was performed to obtain R26 MER/MER MEFs that co-expressed shRNA p53 and miR-92. In this experiment, MEFs were initially infected with an ecotropic MSCV-p53shRNA-SV40huCD4 retrovirus to a nearly 100% infection efficiency, as validated by FACS analysis using huCD4 antibody. The second infection was achieved using an amphotropic MSCV-miR-92-PGK-Puro-IRES-GFP retrovirus. Doubly infected cells were then selected using puromycin. In Figure 5H, double infection of R26 MER/MER MEFs with pRetroX-fbw7-IRES-DsRedExpress and MSCV-miR-92-PGK-Puro-IRES-GFP were similarly performed. For all experiments with primary murine B-cells, bone marrow cells were cultured for 48 hr before retroviral infection and collected or analyzed 72 hr after infection. After 5 days in culture, the percentage of B220-positive cell is 100%. In Figure 3E, Figure 3-figure supplement 1, B-cells were infected with MSCV retrovirus containing a PGK-Puro-IRES-GFP cassette. FACS analysis was performed after gating on the GFP-positive population. In Figure 5A, the collected B-cells were infected with retrovirus containing SV40-CD4 cassette. Infected cells were purified with Human CD4 Miltenyi Biotec).

The collection of normal and malignant B-cells in vivo
Normal mouse B-cells were isolated from the spleen or the bone marrow of 4-to 6-week-old C57B/6J mice, using CD19 Micro-Beads (Miltenyi Biotec) or by negative selection (Easysep 19754;STEMCELL). Similarly, premalignant Eμ-myc B-cells were extracted from the bone marrow of 5-to 6-week-old Eμmyc transgenic mice. Malignant Eμ-myc B-cells were extracted from the lymph node tumors of terminalstage Eμ-myc mice. In addition, the normal human B-cells from peripheral blood were FACS sorted from the peripheral blood of healthy donors.

Histopathology and immunotyping
Mouse tissue samples were fixed in formalin (SF100-4; Fisher), embedded in paraffin (AC41677-0020; Fisher), sectioned into 5 μm tissue samples, and stained with hematoxylin and eosin (7211 & 7111, Fisher). For caspase-3 (AF835, 1:200; R&D Systems), PCNA (MS-106P, 1:200; Lab Vision Corp.), and B220 (14-0452-85, 1:100; eBioscience) detection, representative sections were deparaffinized and rehydrated in graded alcohols before subjected to antigen retrieval treatment with 10 mM sodium citrate buffer 10 min in a pressure cooker. Detection of antibody staining was carried out following standard procedures from the avidin-biotin immunoperoxidase methods. Diaminobenzidine (002014, Invitrogen) was used as the chromogen and hematoxylin as the nuclear counter stain. Quantitation of apoptosis was evaluated by counting the number of starry sky foci in three fields (40X) from seven representative animals of each genotype, as well as by counting the number of caspase-3 positive cells in three fields (40X) from five representative animals of each genotype.

Apoptosis assays and proliferation assays
Subconfluent MSCV-or miR-92-infected R26 MER/MER MEFs were induced and serum starved by incubating the cells with 100 nM of 4-hydroxytamoxifen (H6278; Sigma) in DMEM with 0.2% fetal bovine serum for 12-24 hr before harvesting the cells for apoptosis analyses using APC-Annexin V antibody (550475, 1:50; BD Pharmingen) and 7AAD staining solution (559925; BD Pharmingen). To evaluate the apoptotic effects of miR-92 in our adoptive transfer model in vivo, we collected premalignant Eμ-myc B-cells from spleen or bone marrow of well-controlled Eμ-myc/92 and Eμ-myc/MSCV mice at 5 weeks after adoptive transfer and measured the extent of apoptosis by FACS. Apoptosis in GFP-positive B220-positive premalignant B-cells was measured using the Caspase Detection Kit (Calbiochem, Red-VAD-FMK) following the manufacturer's instructions. To quantitate cell proliferation, 10 μM of BrdU was used to label primary B-cells for 4 hr and MEFs for 30 min. The percentage of BrdU-positive cells was determined using the Flow BrdU kit (552598; BD Pharmingen).

Microarray analyses
Three independent R26 MER/MER MEF lines were infected by MSCV vector alone or by MSCV vector encoding miR-92. These MEFs were induced and serum starved by incubating the cells with 100 nM of 4-hydroxytamoxifen (H6278; Sigma) in DMEM with 0.2% fetal bovine serum for 12 hr before harvesting the cells for RNA preparation. Total RNAs were prepared using Trizol (15596018; Invitrogen), and subjected to microarray analysis using Affymetrix chip Mouse 430_2. To identify differentially expressed genes that could be regulated by miR-92, we used gcRMA in the bioconductor package (Wu et al., 2004) and SAM (Significance Analysis of Microarrays) (Tusher et al., 2001) for statistical analysis of our microarray data. Gene expression signals were estimated from the probe signal values in the CEL files using statistical algorithm gcRMA. This data processing at the probe level includes background signal subtraction and quantile normalization to facilitate the comparison among microarrays. SAM was then used to identify the genes with significant expression level alterations between miR-92 overexpressing MEFs and the control MEFs. The genes with at least 1.5-fold expression level change and FDR <1% were regarded as differentially expressed genes. Pathway analyses were performed on upregulated and downregulated genes using the KEGG database (Dennis et al., 2003).

Xenopus embryo apoptosis assays
Xenopus laevis eggs were collected, fertilized, and embryos cultured by standard procedures. The miR-19b mimics were produced from the annealing products of 5′UGUGCAAAUCCAUGCAAAACUGA3′ and 5′AGUUUUGCAGGUUUGCAUCCAUU3′ (IDT).
The annealing of miRNA mimics were performed by combining two complimentary RNA oligos at a stock concentration of 1 μg/μl, heating the oligos to 80°C for 1 min, and then cooling down to room temperature to allow duplexes to form. The same was done for generating the mutated miR-19 mimics , by annealing 5′UCAGGUAAUCCAUGCAAAACUGA3′ and 5′AGUUUUGCAGGUUACCU UCGAUU3′, and mutated miR-92 mimics (Mut-miR-92) by annealing 5′UUAUCGACUUGUCCCGG3′ and 5′GGUUGGGAUUGGUUCGA 3′.
Xenopus embryos were injected into both cells at the two-cell stage with 2 ng of each RNA (Walker and Harland, 2009). The pcDNA3-myc-AGO2 vector, kindly provided by Dr Greg Hannon, was cut using ScaI; and the synthetic hAGO2 mRNAs were transcribed using mMessage mMachine T7 kit (Ambion). When indicated, a total of 0.5 ng hAGO2 mRNA (Liu et al., 2004) was injected into two-cell stage embryos either alone or with 2 ng of each miRNA (Lund et al., 2011). The embryos were then treated with hydroxyurea (H8627; Sigma) at a final concentration of 5 mM from stage 3 until stage 10. Apoptotic embryos were scored as those containing any apoptotic cells based on morphological changes.

Luciferase assays
A luciferase reporter fused with the fbw7 3′UTR was kindly provided by Dr Hans-Guido Wendel (Mavrakis et al., 2011). In this psiCHECK-2 based reporter, the fbw7 3′UTR was cloned downstream of the Renilla luciferase reporter, and a separate firefly luciferase cassette was used as a transfection control. Because the two predicted miR-92 binding sites are close to each end of the 3′UTR, we mutated the miR-92 binding sites by PCR using the following primers: 3′UTR-Fbw7-Mut-Xho1-F (GATCTCGAGCAAGACGACTCTCTAAATCCAACTATTCTTT) and 3′UTR-Fbw7-mut-Not1-R (ATGCGGCCGCAACACATTTAGTTATAAGAAAATAAAATTT). The PCR fragment was subsequently cloned into the XhoI and Not1 sites of the psiCHECK-2 vector. The reporter construct, together with 50 nM miR-92 mimics, was transfected into Dicer-deficient Hct116 cells (Cummins et al., 2006), with transfection of miR-17 or miR-18 as negative controls. Luciferase activity of each construct was determined by dual luciferase assay (E19100; Promega) 48 hr post-transfection following the manufacturer's instructions. The miR-17 mimics were produced by annealing 5′CAAAGUGCUUAC AGUGCAGGUAGU3′ and 5′UACUGCAGUGAAGGCACUUGUAG3′. The miR-18 mimics were produced by annealing 5′UAAGGUGCAUCUAGUGCAGAUAG3′ and 5′ACUGCCCUAAGUGCUCC UUCUG3′.

Fbw7α immunoprecipitation and western analyses
Because Fbxw7-substrate degradation was regulated in a cell-cycle-dependent manner, we used serum starvation synchronized MEFs to study Fbw7 regulation by miR-92 during cell cycle progression. MEFs were made quiescent by serum starvation; then Fbw7 expression was examined following release into serum. Cells were lysed in NP-40 buffer supplemented with protease inhibitors. Lysates were normalized and immunoprecipitated with polyclonal anti-Fbw7 antibody kindly provided by Dr Bruce Clurman (Grim et al., 2008), followed by immunoblotting with polyclonal anti-Fbw7 antibody (A301-720A; Bethyl Laboratories). Wild-type and FBW7 −/− Hct116 cells were used, respectively, as positive and negative controls.
The construction of the pFLAG-Fbw7α-3′UTR plasmid was previously described (Xu et al., 2010). The construct was transfected into the Dicer-deficient Hct116 cells together with 50 nM of miR-92 mimics or siRNA against GFP as indicated. Anti-FLAG (M2; Sigma) antibody was used to detect the FLAG-Fbw7α by western blot 48 hr after transfection.

Research article
Cyclin E-dependent kinase assays Cyclin E-CDK complexes were immunoprecipitated from MSCV or miR-92 infected Rosa26 MER/MER MEFs extracts using affinity-purified polyclonal antibody, provided by Dr Bruce Clurman (Minella et al., 2008). Cyclin E immunoprecipitates were then incubated with purified histone subunit H1 (Sigma) and (gamma-32 P)ATP to measure cyclin E-dependent kinase activity. The anti-Grb2 monoclonal (BD Biosciences) antibody was used a normalization control.
Committee (ACUC). The animal protocol detailing the experimental procedures with laboratory mice was carefully reviewed and approved by Animal Care and Use Committee (ACUC) at the University of California at Berkeley. Our Animal Use protocol number is R316-0613BR.