Distinct molecular pathways mediate Mycn and Myc-regulated miR-17-92 microRNA action in Feingold syndrome mouse models

Mirzamohammadi, Fatemeh; Kozlova, Anastasia; Papaioannou, Garyfallia; Paltrinieri, Elena; Ayturk, Ugur M.; Kobayashi, Tatsuya

doi:10.1038/s41467-018-03788-7

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Published: 10 April 2018

Distinct molecular pathways mediate Mycn and Myc-regulated miR-17-92 microRNA action in Feingold syndrome mouse models

Fatemeh Mirzamohammadi¹,
Anastasia Kozlova¹,
Garyfallia Papaioannou¹,
Elena Paltrinieri¹,
Ugur M. Ayturk² &
…
Tatsuya Kobayashi¹

Nature Communications volume 9, Article number: 1352 (2018) Cite this article

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Abstract

Feingold syndrome is a skeletal dysplasia caused by loss-of-function mutations of either MYCN (type 1) or MIR17HG that encodes miR-17-92 microRNAs (type 2). Since miR-17-92 expression is transcriptionally regulated by MYC transcription factors, it has been postulated that Feingold syndrome type 1 and 2 may be caused by a common molecular mechanism. Here we show that Mir17-92 deficiency upregulates TGF-β signaling, whereas Mycn-deficiency downregulates PI3K signaling in limb mesenchymal cells. Genetic or pharmacological inhibition of TGF-β signaling efficiently rescues the skeletal defects caused by Mir17-92 deficiency, suggesting that upregulation of TGF-β signaling is responsible for the skeletal defect of Feingold syndrome type 2. By contrast, the skeletal phenotype of Mycn-deficiency is partially rescued by Pten heterozygosity, but not by TGF-β inhibition. These results strongly suggest that despite the phenotypical similarity, distinct molecular mechanisms underlie the pathoetiology for Feingold syndrome type 1 and 2.

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Introduction

Heterozygous mutations in MYCN or MIR17HG in humans cause Feingold syndrome that is characterized by skeletal developmental defects including microcephaly, short stature, and brachysyndactyly with diminished middle phalanxes^1,2,3. MYCN, a member of the MYC family proto-oncogenes, encodes a transcription factor that regulates genes that promote cell growth and proliferation⁴. Previous studies have shown that MYC (Myc in mice) transcription factors bind directly to the MIR17HG gene (Mirc1, also known as Mir17-92 in mice) to stimulate the expression of miR-17-92 microRNAs (miRNAs) encoded by this gene^{5, 6}.

miR-17-92 cluster miRNAs are oncogenic miRNAs that have been studied mainly in the context of cancers^{7, 8}. miR-17-92 miRNAs regulate genes involved in cell cycle and apoptosis, such as Bim (Bcll11)^{9, 10} and E2f1⁵, as well as target genes encoding signaling molecules^{9, 11,12,13} in a context-dependent manner to promote tumorigenesis.

miR-17-92 miRNA family miRNAs, comprised of four functionally overlapping groups of miRNAs, are also encoded by two additional paralogous genes, MIR106B (Mirc3, also known as Mir106b-25 in mice) and MIR106A (Mirc2, also known as Mir106a-363 in mice). The physiologic role of miR-17-92 family miRNAs during animal development was demonstrated by genetic mutant mice missing these genes¹⁰. Homozygous deletion of Mir17-92 in mice impairs animal growth and causes prenatal lethality. Additional deletion of Mir106b-25 results in significantly more severe developmental defects, providing genetic evidence for the overlapping function of these genes.

Based on the fact that deletion of either MYCN or Mir17HG results in similar skeletal defects in humans and that miR-17-92 expression is regulated by Myc transcription factors, it has been hypothesized that Mycn and miR-17-92 miRNAs function in the same pathway to regulate skeletal development². To test this hypothesis, we conditionally deleted Mir17-92 with or without deletion of Mir106b-25 in the developing limb bud and skull mesenchyme.

In this study, we present evidence that Mycn and miR-17-92 miRNAs regulate skeletal progenitor cell proliferation through distinct signaling pathways. We show that overactivation of TGF-β signaling causes the skeletal defects of miR-17-92 miRNA-deficient limbs and the skull, whereas downregulation of PI3K/Akt signaling is a major contributor to the skeletal phenotype caused by Mycn-deficiency. This study using in vivo models also suggests that physiological effects of miRNAs and transcription factors, which regulate multiple genes, converge onto relatively limited signaling pathways.

Results

Generation of mouse models of Feingold syndrome patients

In order to determine physiologic roles of miR-17-92 miRNAs in skeletal development, we deleted Mir17-92 with or without its paralogous gene, Mir106b-25, in skeletal mesenchymal cells using Prx1-Cre transgenic mice and floxed Mir17-92 and Mir106b-25-null mice. Mice with heterozygous Mir17-92 deletion (Prx1-Cre:Mir17-92^fl/+) exhibited modest skeletal defects similar to those in germline heterozygous null mice² (Supplementary Fig. 1a-d). The skeletal phenotype was more pronounced after deletion of both alleles of Mir17-92 (Fig. 1; Supplementary Fig. 1a-d). Simultaneous deletion of Mir17-92 and Mir106b-25 (doubly conditional knockout, Prx1-Cre:Mir17-92^fl/fl:Mir106b-25^−/−, 17 dKO) caused microcephaly, cutaneous syndactyly, and brachydactyly, phenotypes similar to those observed in patients with Feingold syndrome type 2 (Fig. 1a–d). While heterozygous Mycn germline deletion in mice does not cause detectable skeletal abnormalities¹⁴, conditional deletion of Mycn in developing limbs (Prx1-Cre:Mycn^fl/fl, Mycn cKO) resulted in severe brachysyndactyly similar to that observed in patients with Feingold syndrome type 1 and also in mice previously reported¹⁵ (Fig. 1a–d).

Mycn-deficient limb mesenchymal cells isolated from embryos at indicated embryonic days (E) showed significant reductions in miR-17-92 miRNA levels over time, as assessed by quantitative RT-PCR (qRT-PCR) and in situ hybridization (Fig. 1e; Supplementary Fig. 2). Thus, we attempted to rescue the skeletal phenotype of Mycn-deficient limbs by overexpressing miR-17-92 miRNAs using Cre-inducible Mir17-92 transgenic mice⁹. Mir17-92 overexpression in transgenic mice (Prx1-Cre:Mir17-92^Tg, 17 Tg) caused limb overgrowth (Supplementary Fig. 3), and it partially rescued the skeletal abnormalities of Mycn cKO mice (Fig. 1f). These results are consistent with the notion that the action of Mycn is mediated, at least in part, by miR-17-92 miRNAs in skeletal development.

Proliferation defects in miR-17-92 miRNA-deficient cells

To assess the biological consequence of loss of Mir17-92 and Mir106b-25 in the skeletal mesenchyme, we measured cell proliferation and apoptosis in the skull and limbs of mutant embryos. Simultaneous loss of Mir17-92 and Mir106b-25 in the early limb bud and skull mesenchyme caused a significant decrease in cell proliferation (Fig. 2a–c), which was also confirmed in vitro (Fig. 2d). We did not find significant changes in cell death assessed by the TUNEL assay (Supplementary Fig. 4a). The domain expressing Sox9, a marker of precartilaginous condensations, was reduced in size in 17 dKO mice at E9.5 and E11.5, presumably due to the proliferation defect of 17 dKO limb mesenchymal cells (Fig. 2e). Conditional deletion of Mir17-92 and Mir106b-25 exclusively in the cells of the precartilaginous condensation and their descendants using Col2a1-Cre transgenic mice caused no obvious skeletal abnormalities (Supplementary Fig. 4b), suggesting that the proliferation defect in cells of the limb bud mesenchyme, but not cells in the precartilaginous condensation, is responsible for the skeletal abnormalities of 17 dKO mice. Since Mycn deficiency also reduces limb bud cell proliferation¹⁵, these findings suggest that similar cellular mechanisms underlie the pathogenesis of Feingold syndrome type 1 and type 2.

TGF-β and STAT3 signaling upregulation in Mir17-92-deficiency

Although a miRNA potentially regulates hundreds of target genes, it is proposed that miRNA–RNA interactions are evolutionarily selected to cooperatively regulate certain biological pathways^16,17,18. In addition to directly regulating genes involved in cell cycle and apoptosis^{5, 9}, miR-17-92 miRNAs are reported to regulate genes encoding signaling molecules in a context-dependent manner to promote tumorigenesis^{11, 13, 19}. Therefore, we first determined the status of major signaling pathways in primary limb or skull mesenchymal cells missing Mir17-92 and Mir106b-25. At the basal culture condition, we found upregulation in TGF-β and JAK/STAT3 signaling (Fig. 3a, b). In addition, Mir17-92:Mir106b-25-deficient cells showed a greater response in Smad2 phosphorylation than control after acute stimulation with TGF-β1 (Fig. 3c). The type II TGF-β receptor, Tgfbr2, a predicted target of miR-17 and miR-19 group miRNAs¹⁹, was upregulated at both the protein and mRNA levels (Fig. 3a, d). Since loss of miR-17 group miRNAs (miR-17 and miR-20a) causes skeletal defects in mice²⁰, we tested whether miR-17 can suppress gene expression via the predicted target sequence of Tgfbr2 by luciferase reporter assay. miR-17 co-transfection significantly reduced the expression of a luciferase reporter construct with the wildtype, but not with a mutated, binding sequence (Fig. 3e). Although Bim and Pten were previously identified as critical miR-17-92 targets in lymphogenesis and tumorigenesis^{9, 10}, we did not find upregulation in Bim or Pten in 17 dKO mesenchymal cells (Fig. 3d), supporting the notion that the regulatory role of miR-17-92 miRNAs is dependent on the cellular context.

Causal role of TGF-β signaling in cell proliferation defect

To test whether the upregulation of the TGF-β or JAK/STAT3 signaling was responsible for the proliferation defect observed in 17 dKO cells, we inhibited these signaling pathways using pathway-specific inhibitors in vitro. Whereas treatment with the STAT3 inhibitor, S31-201, failed to promote proliferation of 17 dKO cells in vitro (Fig. 4a, b), treatment with the TGF-β receptor inhibitor, LY364947, significantly ameliorated the proliferation defect of 17 dKO cells (Fig. 4c, d). This finding was confirmed by experiments using GW788388, another TGF-β receptor inhibitor, and also using the neutralizing antibody against TGF-β ligands, 1D11 (Supplementary Fig. 5a, b). These results suggest that the TGF-β upregulation is responsible for the proliferation defect, a finding consistent with the previous reports that overactivation of TGF-β signaling reduces limb mesenchymal cell proliferation and inhibits limb growth^{21, 22}.

TGF-β inhibition rescues skeletal defects of 17 dKO mice

We treated 17 dKO mice with TGF-β receptor inhibitors from E9.5 through postnatal day (P) 16.5. 17 dKO mice treated with LY364947 showed significant normalization of limb and skull development (Fig. 4e–h). Moreover, genetic suppression of TGF-β signaling via conditionally deleting one allele of Tgfbr2 in 17 dKO mice (17 dKO:Tgfbr2 Het) also partially rescued the skeletal abnormalities of 17 dKO mice (Supplementary Fig. 5c). A short-term treatment regimen during early stages of limb development (E9.5–E15.5) with TGF-β receptor inhibitor mostly rescued the limb and skull abnormalities whereas treatment during late embryonic stages (E15.5–E18.5) showed no effect (Supplementary Fig. 5d). These data suggest that miR-17-92 miRNA-mediated suppression of TGF-β signaling through Tgfbr2 downregulation is particularly important during early stages of skeletal development and that the TGF-β signaling upregulation is responsible for skeletal defects in Feingold syndrome type 2. TGF-β inhibitor treatment also rescued the digit phenotype of mice with heterozygous Mir17-92 deletion, a genetic alteration close to that of patients with Feingold syndrome type 2, further supporting this notion (Supplementary Fig. 6a).

Distinct molecular mechanisms in 17 dKO and Mycn cKO mice

In contrast to 17 dKO mice, suppression of TGF-β signaling via Tgfbr2 heterozygous deletion or TGF-β receptor inhibitor treatment failed to rescue the skeletal defects of Mycn cKO mice (Fig. 5a, b; Supplementary Fig. 6b). Consistent with this result, we did not find upregulation in TGF-β signaling in Mycn-deficient cells, as indicated by the unaltered level of p-Smad2 (Fig. 5c, d). These results conflict with the previously proposed model in which miR-17-92 miRNAs mediate Mycn’s action², and are also inconsistent with our earlier observations that miR-17-92 expression is reduced in E14.5 Mycn-deficient limbs and that miR-17-92 overexpression rescues the Mycn cKO digit phenotype (Fig. 1e, f). However, we noted that the reduction of miR-17-92 miRNA expression levels in Mycn cKO cells was relatively modest at E10.5 when TGF-β suppression by miR-17-92 miRNAs is critical to maintain limb mesenchymal cell proliferation (Fig. 1e). Consistent with this finding, known miR-17-92 miRNAs target genes, including Tgfbr2, were not deregulated in Mycn-deficient cells at E10.5 (Fig. 5d). We compared the gene expression profiles of Mycn-deficient and 17 dKO limb mesenchymal cells by RNA-Seq, but found an only limited overlap in genes whose expression was altered in these two groups (Supplementary Fig. 7a; Supplementary Data 1). Together, these data demonstrate that, unlike in the model of Feingold syndrome type 2, TGF-β deregulation does not play a causal role in the pathogenesis of the type 1 Feingold syndrome model (Mycn deletion).

Role of PI3K downregulation in Mycn cKO mice

To further investigate the molecular mechanism for the type 1 model, we revisited the fact that miR-17-92 overexpression rescued the Mycn cKO digit phenotype. The miR-17-92 miRNA levels in the limb mesenchymal cells of 17 Tg mice were four times more abundant than those in control (Supplementary Fig. 8a). We also found that these supraphysiological levels of miR-17-92 miRNAs suppressed the expression of Pten, a negative regulator of phosphoinositide 3-kinase (PI3K)/Akt signaling, and upregulated the phospho-Akt (p-Akt) level (Supplementary Fig. 8b, c). Thus, we hypothesized that deregulation of the PI3K/Akt signaling pathway was involved in the pathogenesis of the Mycn cKO phenotype. Indeed, we found that Mycn-deficient mesenchymal cells showed a decreased level of p-Akt (Thr308), suggesting downregulation of the PI3K signaling pathway (Fig. 5c).

To test whether the suppression of the PI3K signaling pathway is responsible for the skeletal abnormalities in Mycn cKO, we deleted one allele of Pten in Mycn cKO mice (Mycn cKO:Pten Het). Although Pten haploinsufficiency itself did not cause any skeletal phenotype, we found that Pten heterozygous deletion in Mycn-deficient skeletal progenitor cells partially rescued the skeletal abnormality of Mycn cKO mice to a similar extent to Mir17-92 overexpression (Mycn cKO: 17 Tg) (Fig. 6a, b). We confirmed that the p-Akt level increased in Mycn cKO limb bud cells after deletion of one allele of Pten (Fig. 6c). In contrast, Pten heterozygous deletion showed no effect on the 17 dKO skeletal phenotype (Supplementary Fig. 8d). These data suggest that downregulation of the PI3K signaling pathway plays a major pathogenic role in the Feingold syndrome type 1, but not type 2.

Regarding the mechanism by which Mycn deficiency reduces PI3K signaling, there were no significant changes in the expression level of genes related to the PI3K complex, Pten, or their downstream effectors in Mycn cKO limb bud cells (Supplementary Fig. 7b). However, we found decreased phosphorylation of the tail region of Pten (p-Pten) that negatively regulates Pten activity²³ (Fig. 6d). Casein kinase 2 (CK2) is a major regulator of Pten phosphorylation and activity^24,25,26, and CK2 and Myc have been shown to synergistically promote oncogenesis via the CK2’s promoting action on Myc stabilization²⁷. Therefore, we examined the expression and activation of CK2. While the total expression level of CK2 genes was unaltered (Supplementary Fig. 7c, d), CK2 phosphorylation, which reflects CK2 activity²⁸, was reduced in Mycn-deficient cells (Fig. 6e). These results suggest that the downregulation of the CK2/Pten cascade is responsible, at least in part, for the reduced PI3K signaling caused by Mycn deficiency.

Discussion

In this study, we generated mouse models that exhibited skeletal phenotypes similar to those in patients with Feingold syndrome type 1 and type 2 by conditionally deleting Mycn and Mir17-92, respectively, in skeletal progenitors. Our findings strongly suggest that TGF-β signaling upregulation in mesenchymal progenitor cells plays a major causal role in the skeletal abnormalities of Feingold syndrome type 2, whereas downregulation of the PI3K signaling pathway significantly contributes to the pathogenesis of Feingold syndrome type 1 (Fig. 6f).

Although these mouse models are not exact genetic equivalents of human patients with heterozygous deletions of these genes, considering that heterozygous germline-null mice do not fully recapitulate the phenotypes of patients with Feingold syndrome type 1 and type 2 and that our mouse models recapitulate well the skeletal abnormalities of patients, our mouse models likely share the same pathophysiological mechanisms of these diseases in humans. In addition, the finding that TGF-β inhibition was able to rescue the digit defect (shortening of the middle phalanx) of heterozygous Mir17-92 conditional deletion also supports that the TGF-β deregulation underlies the pathogenesis of Feingold syndrome type 2.

Considering that the cellular mechanism that accounts for the skeletal defects, i.e., reduced proliferation of mesenchymal progenitors, appears to be common in Mir17-92:106b-25-deficient (present study) and Mycn-deficient limbs and skulls¹⁵ and that the well-established molecular relationship between Myc transcription factors and miR-17-92 miRNAs, our finding that deficiencies of Mycn and miR-17-92 miRNAs causes aberrations in distinct signaling pathways was unexpected. As previous study showed², we also found partial reductions in miR-17-92 miRNAs in Mycn-deficient limbs. Considering that Mycn-deficient limbs show a skeletal phenotype more severe than that of Mir17-92:106b-25 doubly conditional nullizygotes, the partial reduction of miR17-92 miRNAs in Mycn-deficient limbs may have only limited biological effects.

In the mouse model for Feingold syndrome type 2, using genetic and pharmacological interventions, we have demonstrated that the upregulation in TGF-β signaling due to derepression of Tgfbr2, a direct target of multiple miR-17-92 miRNAs, plays the causal role. The fact that suppression of TGF-β alone was able to almost completely rescue the skeletal defects of Mir-17-92:106b-25-deficient limbs and skulls indicates that Tgfbr2 is the most physiologically important target of miR-17-92 miRNAs in the context of skeletal development, despite the fact that miR-17-92 miRNAs regulates many other genes in diverse types of cells²⁹. Deletion of the endogenous miR-17-92 miRNAs showed no effects on the expression of other known targets, including Pten. However, overexpression of miR-17-92 miRNAs to a supraphysiological level did suppress Pten. This result suggests that suppressive effects of miR-17-92 miRNAs on their targets are dependent on the cellular context, absolute expression level of miRNAs, and perhaps miRNA-target RNA stoichiometry.

We have shown that the downregulation of PI3K/Akt signaling contributes to the skeletal phenotype of Mycn cKO mice. Myc transcription factors control cell growth and proliferation downstream of diverse signaling stimuli³⁰; however, little is known how Myc transcription factors regulate signaling pathways. We have found that Mycn-deficiency decreases Pten phosphorylation and CK2 activation, suggesting the alteration of the CK2/Pten pathway is likely the cause for the reduced PI3K/Akt signaling in Mycn-deficient cells. The precise mechanism by which Mycn deficiency alters the CK2/Pten pathway is unclear at the moment.

In summary, we have demonstrated that distinct molecular mechanisms are responsible for skeletal phenotypes of mouse models of Feingold syndrome type 1 and type 2. Our results also suggest that targeting specific signaling pathways is a valid therapeutic approach to Feingold syndrome.

Methods

Mice

Floxed Mir17-92 mice and Mir106b-25-null mice¹⁰, Cre-inducible CAG-Mir17-92 transgenic mice⁹, Prx1Cre transgenic mice³¹, Col2Cre transgenic mice³², and floxed Mycn mice³³ were previously described. Mice are in a C57/B6-dominant mixed background. Mutant mice were compared with littermate control mice except mice treated with chemical inhibitors during pregnancy. Since both males and females show the same phenotype with similar degrees, males and females were not discriminated. The mutant mice generally showed qualitative phenotypes; more than three independent experiments were performed to ensure the reproducibility. Due to the nature of experiments, no randomization or blinding was used.

The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Massachusetts General Hospital and performed in accordance with the regulations and guidelines.

Reagents

Recombinant mouse TGF-β1 (#5231) was purchased from Cell Signaling Technology. Ly364947 (#S2805), GW788388 (#S2750), and S3I-201 (#S1155) were purchased from Selleck Chemicals.

TGF-β receptor inhibitor injection

TGF-β receptor inhibitors, Ly364947 and GW788388, were dissolved in DMSO at the concentration of 1 mg/ml and 2 mg/ml, respectively. Ly364947 (1 mg/kg/day) and GW788388 (1 mg/kg/day) were injected intraperitoneally (i.p.) into pregnant mothers or nursing mothers until P7.5. Mice older than P7.5 were individually injected.

Skeletal preparation and histology

Alizarin red and alcian blue staining was performed using a modified McLeod’s method³⁴.

For histological analysis, the embryos were dissected, fixed in 10% formalin, paraffin-processed, cut, and subjected to BrdU staining and TUNEL staining using the In situ Cell Death Detection kit (Sigma-Aldrich).

Cell proliferation assays

For BrdU labeling, BrdU (50 mg/kg) was injected into pregnant mice i.p. 2 h before euthanasia. BrdU was detected using the BrdU in situ staining kit (Invitrogen). The BrdU labeling index was calculated as the ratio of BrdU-positive stained nuclei over total nuclei. In vitro cell proliferation was quantified using the PrestoBlue Cell Viability Reagent (Molecular Probes) according to the manufacturer’s instruction.

Primary limb bud cells isolation and culture

Isolation and culture of primary limb bud cells of E9.5 through E11.5 mice were performed as previously described³⁵. Briefly, embryos were isolated from timed-mated females by cesarean section. Limb buds, dissected using fine tweezers, were treated with 0.25% trypsin containing 2.21 mM EDTA for 5 min to disperse cells before plating. After overnight culture, cells were trypsinized and replated at the concentration of 5 × 10⁵/ml in a DMEM medium containing 10% fetal bovine serum (FBS). Upon reaching confluence, cells were collected to assess the basal status of indicated signaling pathways. Cells were also serum-starved for 3 h before stimulating with 10% FBS containing 50 ng/ml of TGF-β1. To assess Smad2 phosphorylation, cells were lysed at indicated time points and subjected to Western blot analysis. For RNA isolation, four limb buds of E10.5 and E11.5 embryos were directly lysed in Trizol. For E14.5 limbs, paws of four limbs were dissected to remove the skin and lysed in Trizol.

For in vitro Mir17-92 deletion, primary limb cells isolated from Mir17-92 homozygous floxed, Mir106b null embryos at E10.5 and were cultured in a DMEM medium containing 10% FBS until reaching 50% confluence. Adenoviruses expressing either Cre or YFP (control) were then added to delete Mir17-92. Then cells were split and grown in 96-well plates for indicated days.

In situ hybridization

Non-radioactive whole-mount in situ hybridization of mouse embryos was done using a standard protocol³⁶. The Sox9 probe was previously described³⁷. Section in situ hybridization for miR-17-5p was performed according to the standard protocol with minor modifications³⁸. E14.5 forelimbs of Prx1-Cre:Mycn^fl/fl mice and Cre-negative littermate control were formalin-fixed, paraffin-processed, cut at 5 µm thickness. The DIG-labeled miR-17 oligo probe (mmu-miR-17-5p; YD00615470-BCG) was purchased from Qiagen.

Adenovirus production and infection

Adenoviruses expressing Cre or YFP were gifts from Dr. Murat Bastepe (Harvard Medical School). Amounts of adenoviruses used for infection were empirically determined to achieve >75% infection efficiency. The infection efficiency was determined 2 days after infection by visualizing YFP for YFP-expressing adenoviruses or by quantifying floxed gene products for Cre-expressing adenoviruses.

Primary embryonic limb bud cell isolation

Prx1Cre: Mycn^fl/+ male and Mycn^fl/fl female mice were mated and mating was confirmed by the presence of a vaginal plug (considered as day 0.5 of gestation). Similarly, Prx1Cre: Mir17-92^fl/+:Mir106b-25^+/− male and Mir17-92^fl/fl:Mir106b-25^−/− female mice were mated to generate doubly conditional knockout mice. On the day 10.5 of gestation, pregnant female mice were sacrificed. The uterus was removed and washed in sterile calcium and magnesium-free PBS. E10.5 embryos were then recovered from the uterus and washed several times in PBS. Distal parts of fore and hind limb buds of embryos were isolated under a dissecting microscope using two pairs of sharp forceps and lysed in TRIzol (Molecular Research Center, Cincinnati, OH, USA). Total RNA was extracted using the Direct-Zol TM RNA MiniPrep kit (Zymo Research, USA) according to the manufacturer’s instruction.

RNA sequencing

RNA-seq libraries were prepared with the Truseq mRNA kit (Illumina Inc., San Diego, CA, USA) with polyA + mRNA selection. Libraries were sequenced with the Illumina NextSeq system with 75 bp single end reads to generate 36–52 million reads per library. Reads were mapped to the mouse genome (mm10) with the STAR aligner³⁹, and differential expression analyses between biologic groups were performed with edger³⁹. The significance level was set at p < 0.05 (after correction for multiple hypothesis testing) for genes with mean FPKM >3 in at least one biologic group.

qRT-PCR

cDNA synthesis was performed using random hexamers using the DyNAmo cDNA Synthesis Kit (Finnzymes). Quantitative PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems) and the EvaGreen qRT-PCR mix (Solix BioDyne). Primer sequences are as follows: Pten-L, 5′-GAAAGGGACGGACTGGTGTA-3′ and Pten-R, 5′-AGTGCCACGGGTCTGTAATC-3′; GAPDH-L, 5′-CACAATTTCCATCCCAGACC-3′ and GAPDH-R, 5′-GTGGGTGCAGCGAACTTTAT-3′; Smad2-L, 5′-AGTATGGACACAGGCTCTCC-3′ and Smad2-R, 5′-GTCTGCCTTCGGTATTCTG-3′; Smad4-L, 5′-ATCTGAGTCTAATGCTACCAGC-3′ and Smad4-R, 5′-TTCTTTGATGCTCTGTCTTGG-3′; Bim-L, 5′-GCCCCTACCTCCCTACAGAC-3′ and Bim-R, 5′- GGCATCACCGTGGATATTTT-3′; Tgfbr2-L, 5′-GCAGTGGGAGAAGTAAAAGA-3′ and Tgfbr2-R, 5′- CCAGCCTGCCCCATAAGAGC-3′. Pik3r1-R, 5′-GCAGCTGAGTACCGAGAGAT-3′; Pik3r1-L, 5′-GCCACTCGTTCAGCTTCTTC-3′; Pik3r3-R, 5′-ACCGAGTACAAGCAGAGGAC-3′; Pik3r3-L, 5′-GCAAAGCCATATCCTCGAGC-3′; Pik3r5-R, 5′-TTTCAGGGAAGGTGGCTAGG-3′; Pik3r5-L, 5′-GTTCCGTGGCTTCTCTTCAC-3′; Pik3ip1-R, 5′-CAAAGAGGCACAGGTGTTCC-3′; Pik3ip1-L, 5′-TGTAGCCCACGATAATGCCA-3′; Akt1s1-R, 5′-AAGAGGACAGAAGCCCGATC-3′; Akt1s1-L, 5′-GGAAGTCGCTGGTATTGAGC-3′. Expression of miR-17-92 miRNAs and miR-106b-25 miRNAs were determined using the mirVana qRT-PCR miRNA Detection Kit (Ambion).

Luciferase reporter assay

A 350-bp-long DNA fragment containing the conserved miR-17 binding sequence (5′-GCACTTTA-3′) or a mutated sequence (5′-GCTCTATA-3′) was PCR amplified using primers, Common R, 5′-GCAAGCTTCTCAGCTCCCTGGTCCATAA-3′ and Wt-F, 5′-GCACTAGTGATCAGCTAATTGACCAGATGCACTTTATTAATGCCTGTGTGTAAATACGAA-3′ or Mut-F, 5′-GCACTAGTGATCAGCTAATTGACCAGATGCTCTATATTAATGCCTGTGTGTAAATACGAA-3′ and subcloned into pMIR-REPORT miRNA Expression Reporter Vector System (AM5795, ThermoFisher) at the Hind III and Spe I sites. miR-17 miRIDIAN microRNA mimic and a control mimic were purchased from Dharmacon. A renilla luciferase expression construct, a Tgfbr2-UTR reporter construct, and the miR-17 mimic or control mimic were co-transfected to primary limb bud cells isolated from E12.5 embryos using Attractene transfection reagent (Qiagen) according to manufacturer’s instruction. Forty-eight hours after transfection, the cells were lysed, and luciferase and renilla luciferase activities were measured using the Dual-luciferase reporter assay system (Promega).

Western blot analysis

Anti-Smad2 (#5339), p-Smad2 (Ser465/467 #3108), p-Akt (Thr308, #2965), p-Akt (Ser473, #4060), NFkB (#3037), p-Erk (Ser259 #9911), p-p38 (#4511), p-Jnk (#4668), p-Stat3 (#9145), p-Stat1 (#9171), Tgfbr2 (#3713), p-IRS1 (Tyr895, 3070), p-Smad1/5(#9511), p-Pten (Ser380/Thr382/383, #9549), Pten (D4.3 #9188), and Act-b (13E5, #4970) antibodies were purchased from Cell Signaling Technology. Anti-p-Akt (T308) antibody (# 658320) was purchased from R&D Systems. Anti-Mycn (B8.4.B, #sc-53993) antibody was purchased from Santa Cruz Biotechnology. Anti-pCKIIβ (S209) antibody (STJ90892) was purchased from St Joh’s laboratory. Anti-Gapdh antibody (10R-2932) was purchased from Fitzgerald Industries International. Western blot analysis was performed according to the standard procedure. Uncropped blots are included in Supplemetary Figs. 9–12.

Data availability

The RNA-seq data are uploaded to the ArrayExpress database with the accession number, E-MTAB-5671 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5671/).

Statistics

Data are presented as mean ± SEM from indicated numbers of samples. For statistical analysis between the two groups, we used a 2-tailed Student’s t test; p values less than 0.05 were considered statistically significant. Sample numbers were determined based on the effect size obtained during preliminary experiments to obtain a power of 0.8.

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Acknowledgements

This work is supported by the US NIH grant, AR056645 (TK). We thank Center for Skeletal Research (NIH P30 AR066261) for histological assistance. We thank Henry Kronenberg, Marc Wein, and Matthew Warman for valuable comments and suggestions.

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Authors and Affiliations

Endocrine Unit, Massachusetts General Hospital, and Harvard Medical School, Boston, 02114, MA, USA
Fatemeh Mirzamohammadi, Anastasia Kozlova, Garyfallia Papaioannou, Elena Paltrinieri & Tatsuya Kobayashi
Musculoskeletal Integrity Program, Hospital for Special Surgery, New York, 10021, NY, USA
Ugur M. Ayturk

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Fatemeh Mirzamohammadi
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Anastasia Kozlova
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Garyfallia Papaioannou
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Elena Paltrinieri
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Ugur M. Ayturk
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Tatsuya Kobayashi
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Contributions

F.M. and T.K. conceived the project and designed the experiments. F.M., A.K., E.P., G.P., U.M.A., and T.K. performed the experiments and interpreted the results. F.M., A.K., and T.K. wrote the manuscript.

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Correspondence to Tatsuya Kobayashi.

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Mirzamohammadi, F., Kozlova, A., Papaioannou, G. et al. Distinct molecular pathways mediate Mycn and Myc-regulated miR-17-92 microRNA action in Feingold syndrome mouse models. Nat Commun 9, 1352 (2018). https://doi.org/10.1038/s41467-018-03788-7

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Received: 10 July 2017
Accepted: 13 March 2018
Published: 10 April 2018
DOI: https://doi.org/10.1038/s41467-018-03788-7

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