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.

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.

Polycistronic miRNAs often exhibit pleiotropic biological functions with unique gene regulatory mechanisms (Megraw et al., 2007). One of the best example is mir-17-92, a potent oncomir (i.e., miRNA oncogene), whose genomic amplification and aberrant overexpression have been observed in many human tumors including Burkitt’s lymphoma, diffuse large B-cell lymphoma (DLBCL), and lung cancer (Lu et al., 2005; Mendell, 2008). mir-17-92 regulates multiple cellular processes during tumor development, including proliferation, survival, angiogenesis, differentiation, and metastasis (He et al., 2007; Uziel et al., 2009; Conkrite et al., 2011; Nittner et al., 2012). As a polycistronic oncomir, mir-17-92 produces a single precursor that yields six individual mature miRNAs (Figure 1A, Figure1—figure supplement 1A) (Tanzer and Stadler, 2004). Based on the seed sequence homology, the six mir-17-92 components are categorized into four miRNA families (Figure 1A, Figure 1—figure supplement 1A): miR-17 (miR-17 and 20), miR-18, miR-19 (miR-19a and 19b), and miR-92a (we will designate miR-92a as miR-92 in the remainder of our paper). Interestingly, miR-92 has a more ancient evolutionary history compared to the other mir-17-92 components (Tanzer and Stadler, 2004). miR-92 is evolutionarily conserved in vertebrates, chordates, and invertebrates, while the remaining mir-17-92 components are only found in vertebrates (Figure 1—figure supplement 1B,C). Conceivably, the distinct mature miRNA sequence of each mir-17-92 component determines the specificity of the target regulation. However, the functional significance of the mir-17-92 polycistronic gene structure remains largely unknown.

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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 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.

Since mir-17-92 is overexpressed in human Burkitt’s lymphomas (Tagawa et al., 2007), we set out to functionally dissect mir-17-92 components in the Eμ-myc model of Burkitt’s lymphoma (Figure 1B). The Eμ-myc mice carry a c-myc transgene downstream of the immunoglobulin (Ig) heavy chain enhancer Eμ (Langdon, 1986; Adams et al., 1985), which functionally resembles the Ig-MYC translocations that occur frequently in Burkitt’s lymphomas (Tagawa et al., 2007). The resulting B-cell specific, aberrant c-Myc activation promotes excessive proliferation, yet also evokes potent, p53-dependent apoptosis (Schmitt et al., 2002; Hemann et al., 2003). Thus, c-Myc-induced apoptosis enables a self-defense mechanism against malignant transformation, producing B-lymphomas with a late onset (Lowe et al., 2004). In our adoptive transfer model (Olive et al., 2009), Eμ-myc/+ hematopoietic stem and progenitor cells (HSPCs) were transplanted into lethally irradiated recipient mice, generating chimeric mice that faithfully recapitulated the late tumor onset of the Eμ-myc transgenic mice (Figure 1B).

When Eμ-myc/+ HSPCs were infected with MSCV (murine stem cell virus) retrovirus to overexpress the intact mir-17-92 oncomir, we observed a considerable acceleration in tumor onset compared to the Eμ-myc/MSCV control mice (p<0.01, Figure 1C). Unexpectedly, the oncogenic cooperation between c-Myc and mir-17-92 was significantly stronger when miR-92 was deleted within this oncomir (Figure 1C). The average survival of Eμ-myc/17-92Δ92 mice was 66 days, significantly shorter than that of Eμ-myc/17-92 mice (112 days, p<0.0001). mir-17-92Δ92 carried a deletion of miR-92 pre-miRNA and its flanking sequences, which might alter the expression of the remaining mir-17-92 components (Figure 1D, Figure 1—figure supplement 1D). We then engineered a 12-nucleotide miR-92 seed mutation within mir-17-92 to abolish the functional miR-92 with minimal disruption to the overall gene structure. The resulting mir-17-92Mut92 phenocopied mir-17-92Δ92 in vivo (Figure 1C), significantly enhancing the oncogenic cooperation with c-Myc without altering the level of any remaining mir-17-92 components (Figure 1D, Figure 1—figure supplement 1D). This unexpected effect was specifically attributable to miR-92. Mutations of miR-20 or miR-17 failed to affect oncogenesis in the Eμ-myc model (Figure 1C, Figure 1—figure supplement 1D and data not shown), and mutations of both miR-19 miRNAs nearly abolished this oncogenic cooperation (Olive et al., 2009). This finding suggests that, although mir-17-92 acted as a potent oncogene as a whole, its miR-92 component confers an internal negative regulation on its oncogenic cooperation with c-Myc. This effect of miR-92 clearly contrasts with that of miR-19, a key oncogenic mir-17-92 component that promotes c-Myc-induced lymphomagenesis by repressing apoptosis (Mu et al., 2009; Olive et al., 2009; Mavrakis et al., 2010).

In the Eμ-myc model, a strong oncogenic lesion often leads to the B-cell transformation at an earlier developmental stage (Hemann et al., 2003). The greater oncogenic activity of mir-17-92Mut92 in comparison with mir-17-92 was consistent with mir-17-92Mut92 preferentially transforming IgM negative progenitor B-cells, and mir-17-92 frequently transforming IgM positive B-cells (Figure 2A; Table 1). In comparison to Eμ-myc/17-92 mice, both Eμ-myc/17-92Δ92 and Eμ-myc/17-92Mut92 mice developed more aggressive B-lymphomas, characterized by massive lymph node enlargement, splenic hyperplasia, leukemia, and widespread dissemination into visceral organs outside of the lymphoid compartment (Figure 2B, data not shown).

Figure 2

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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 Eμ-myc/17-92, Eμ-myc/17-92Δ92, Eμ-myc/17-92Mut92, 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,B, Figure 3—figure supplement 1A), consistent with a rapid depletion of miR-92-infected cells in premalignant Eμ-myc B-cells (Figure 3—figure supplement 1B). Similar miR-92 effects on c-Myc-induced apoptosis were observed in vitro. The R26^MER/MER mouse embryonic fibroblasts (MEFs) carry a switchable variant of Myc, MycER^T2, downstream of the constitutive Rosa26 promoter, which allows acute activation of the MycER transgene by 4-OHT (4-Hydroxytamoxifen) induced nuclear translocation (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). Enforced miR-92 expression in R26^MER/MER MEFs invariably enhanced Myc-induced apoptosis (Figure 3C, Figure 3—figure supplement 1C).

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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 B-cells revealed that the miR-92 component yielded one of the strongest effects (Figure 3E,F). In addition, miR-92 deficiency significantly compromised the ability of mir-17-92 to promote cell cycle progression in B-cells (Figure 3—figure supplement 1D). Interestingly, strong proliferative effects have been reported for nearly all mir-17-92 components, yet the exact cell type and biological context can select specific components as the predominant drivers for cell proliferation. Taken together, our data suggest that miR-92 is a unique mir-17-92 component that functionally couples c-Myc-induced cell proliferation and c-Myc-induced apoptosis in the B-cell compartment.

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.

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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 Mdm2-mediated 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 4C,D, Figure 4—figure supplement 1C), consistent with the rapid stabilization of the p53 protein (Figure 4D, Figure 4—figure supplement 1C) without alteration of p53 mRNA (Figure 4—figure supplement 1D). Notably, the ability of miR-92 to induce p53 activation occurred not only in 4-OHT treated R26^MER/MER MEFs with MycER^T2 activation, but also in untreated R26^MER/MER MEFs with normal c-Myc level. This was clearly demonstrated by the elevation of p53 protein level, as well as the increased p53 target expression (Figure 4—figure supplement 1B,C).

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-Myc-induced 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.

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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 domain-containing 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, 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 6—figure supplement 1B).

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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, 2005; Miranda 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 6—figure 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 c-myc driven B-lymphomas. In the LT2-MYC murine model of hepatocellular carcinoma (HCC), where tumor development was initiated by tetracycline-inducible c-Myc expression, miR-19a and miR-19b also exhibited a stronger increase than miR-92 when comparing tumor cells and the normal counterpart (Figure 7E).

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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.

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-Myc-induced 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. 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 Ras-induced 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 1—figure 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.

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Author details

1. Virginie Olive

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   VO, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Erich Sabio

   Competing interests

   The authors declare that no competing interests exist.

2. Erich Sabio

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   ES, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Virginie Olive

   Competing interests

   The authors declare that no competing interests exist.

3. Margaux J Bennett

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   MJB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Caitlin S De Jong

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   CSDJ, Conception and design, Acquisition of data

   Competing interests

   The authors declare that no competing interests exist.

5. Anne Biton

   Department of Statistics, University of California, Berkeley, Berkeley, United States

   Contribution

   AB, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

6. James C McGann

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Present address

   Vollum Institute, Oregon Health and Science University, Portland, United States

   Contribution

   JCM, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

7. Samantha K Greaney

   Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

   Contribution

   SKG, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

8. Nicole M Sodir

   Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom

   Contribution

   NMS, Conception and design, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

9. Alicia Y Zhou

   Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

   Contribution

   AYZ, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

10. Asha Balakrishnan

    Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

    Contribution

    AB, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

11. Mona Foth

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

    Contribution

    MF, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

12. Micah A Luftig

    Department of Molecular Genetics and Microbiology, Duke University, Durham, United States

    Contribution

    MAL, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

13. Andrei Goga

    Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

    Contribution

    AG, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents

    Competing interests

    The authors declare that no competing interests exist.

14. Terence P Speed

    Department of Statistics, University of California, Berkeley, Berkeley, United States

    Contribution

    TPS, Conception and design, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

15. Zhenyu Xuan

    Department of Molecular and Cell Biology, Center for Systems Biology, University of Texas at Dallas, Dallas, United States

    Contribution

    ZX, Conception and design, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

16. Gerard I Evan

    Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom

    Contribution

    GIE, Conception and design, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

17. Ying Wan

    Department of Medicine, The Third Military Medical University, Chongqing, China

    Contribution

    YW, Conception and design, Acquisition of data, Analysis and interpretation of data

    Competing interests

    The authors declare that no competing interests exist.

18. Alex C Minella

    Driskill Graduate Program, Department of Medicine, Hematology and Oncology Division, Northwestern University Feinberg School of Medicine, Chicago, United States

    Contribution

    ACM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

19. Lin He

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

    Contribution

    LH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    lhe@berkeley.edu

    Competing interests

    The authors declare that no competing interests exist.

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