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miR-146a promotes the initiation and progression of melanoma by activating Notch signaling.

Forloni M, Dogra SK, Dong Y, Conte D, Ou J, Zhu LJ, Deng A, Mahalingam M, Green MR, Wajapeyee N - Elife (2014)

Bottom Line: We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling.Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164).We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, United States.

ABSTRACT
Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma. DOI: http://dx.doi.org/10.7554/eLife.01460.001.

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Related in: MedlinePlus

Inhibition of MAP kinase signaling by MEK inhibitor blocks HRAS v12-mediated miR-146a upregulation.qRT-PCR analysis (left) to monitor miR-146a expression and immunoblot analysis (right) of phosphorylated (p-) ERK and total (t-) ERK in MEL-ST cells transduced with HRAS v12 in the presence (+) or absence (−) of the MEK inhibitor U0216.DOI:http://dx.doi.org/10.7554/eLife.01460.007
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fig1s4: Inhibition of MAP kinase signaling by MEK inhibitor blocks HRAS v12-mediated miR-146a upregulation.qRT-PCR analysis (left) to monitor miR-146a expression and immunoblot analysis (right) of phosphorylated (p-) ERK and total (t-) ERK in MEL-ST cells transduced with HRAS v12 in the presence (+) or absence (−) of the MEK inhibitor U0216.DOI:http://dx.doi.org/10.7554/eLife.01460.007

Mentions: Next, we asked whether BRAF-MEK-ERK signaling is required for miR-146a upregulation. We stably expressed BRAFV600E or an empty vector in MEL-ST cells, which are immortalized melanocytes that have basal levels of BRAF-MEK-ERK signaling and can be transformed by single oncogenes (Gupta et al., 2005). Figure 1D shows that, as in melanocytes, introduction of BRAFV600E into MEL-ST cells upregulated miR-146a expression. To confirm that miR-146a upregulation requires BRAF-MEK-ERK signaling, we treated MEL-ST/BRAFV600E cells with the MEK inhibitor U0216. Figure 1D shows that treatment of MEL-ST/BRAFV600E cells with U0216 resulted in decreased miR-146a expression. As expected, transcriptional targets of the MAP kinase pathway were also downregulated following U0216 treatment (Figure 1—figure supplement 2). Similar to BRAFV600E, stable expression of NRASQ61K, HRAS v12 or a mutant that selectively activates BRAF-MEK-ERK signaling also resulted in increased miR-146a levels (Figure 1E, Figure 1—figure supplement 3), which was attenuated by addition of U0216 (Figure 1—figure supplement 4).


miR-146a promotes the initiation and progression of melanoma by activating Notch signaling.

Forloni M, Dogra SK, Dong Y, Conte D, Ou J, Zhu LJ, Deng A, Mahalingam M, Green MR, Wajapeyee N - Elife (2014)

Inhibition of MAP kinase signaling by MEK inhibitor blocks HRAS v12-mediated miR-146a upregulation.qRT-PCR analysis (left) to monitor miR-146a expression and immunoblot analysis (right) of phosphorylated (p-) ERK and total (t-) ERK in MEL-ST cells transduced with HRAS v12 in the presence (+) or absence (−) of the MEK inhibitor U0216.DOI:http://dx.doi.org/10.7554/eLife.01460.007
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927633&req=5

fig1s4: Inhibition of MAP kinase signaling by MEK inhibitor blocks HRAS v12-mediated miR-146a upregulation.qRT-PCR analysis (left) to monitor miR-146a expression and immunoblot analysis (right) of phosphorylated (p-) ERK and total (t-) ERK in MEL-ST cells transduced with HRAS v12 in the presence (+) or absence (−) of the MEK inhibitor U0216.DOI:http://dx.doi.org/10.7554/eLife.01460.007
Mentions: Next, we asked whether BRAF-MEK-ERK signaling is required for miR-146a upregulation. We stably expressed BRAFV600E or an empty vector in MEL-ST cells, which are immortalized melanocytes that have basal levels of BRAF-MEK-ERK signaling and can be transformed by single oncogenes (Gupta et al., 2005). Figure 1D shows that, as in melanocytes, introduction of BRAFV600E into MEL-ST cells upregulated miR-146a expression. To confirm that miR-146a upregulation requires BRAF-MEK-ERK signaling, we treated MEL-ST/BRAFV600E cells with the MEK inhibitor U0216. Figure 1D shows that treatment of MEL-ST/BRAFV600E cells with U0216 resulted in decreased miR-146a expression. As expected, transcriptional targets of the MAP kinase pathway were also downregulated following U0216 treatment (Figure 1—figure supplement 2). Similar to BRAFV600E, stable expression of NRASQ61K, HRAS v12 or a mutant that selectively activates BRAF-MEK-ERK signaling also resulted in increased miR-146a levels (Figure 1E, Figure 1—figure supplement 3), which was attenuated by addition of U0216 (Figure 1—figure supplement 4).

Bottom Line: We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling.Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164).We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, United States.

ABSTRACT
Oncogenic mutations in BRAF and NRAS occur in 70% of melanomas. In this study, we identify a microRNA, miR-146a, that is highly upregulated by oncogenic BRAF and NRAS. Expression of miR-146a increases the ability of human melanoma cells to proliferate in culture and form tumors in mice, whereas knockdown of miR-146a has the opposite effects. We show these oncogenic activities are due to miR-146a targeting the NUMB mRNA, a repressor of Notch signaling. Previous studies have shown that pre-miR-146a contains a single nucleotide polymorphism (C>G rs2910164). We find that the ability of pre-miR-146a/G to activate Notch signaling and promote oncogenesis is substantially higher than that of pre-miR-146a/C. Analysis of melanoma cell lines and matched patient samples indicates that during melanoma progression pre-miR-146a/G is enriched relative to pre-miR-146a/C, resulting from a C-to-G somatic mutation in pre-miR-146a/C. Collectively, our results reveal a central role for miR-146a in the initiation and progression of melanoma. DOI: http://dx.doi.org/10.7554/eLife.01460.001.

Show MeSH
Related in: MedlinePlus