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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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pre-miR-146a/G promotes proliferation of melanoma cells more effective than pre-miR-146a/C.M14, SKMEL-28 and A375 cells stably expressing pre-miR-146a/C (blue) or pre-miR-146a/G (red) or an empty vector (black) were analyzed for proliferation at indicated days. Relative proliferation is plotted. *, ** and *** represents p values <0.01, <0.001 and <0.0001 respectively.DOI:http://dx.doi.org/10.7554/eLife.01460.016
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fig3s2: pre-miR-146a/G promotes proliferation of melanoma cells more effective than pre-miR-146a/C.M14, SKMEL-28 and A375 cells stably expressing pre-miR-146a/C (blue) or pre-miR-146a/G (red) or an empty vector (black) were analyzed for proliferation at indicated days. Relative proliferation is plotted. *, ** and *** represents p values <0.01, <0.001 and <0.0001 respectively.DOI:http://dx.doi.org/10.7554/eLife.01460.016

Mentions: Several recent studies have found that a pre-miR-146a SNP (C>G rs2910164) alters the expression of mature miR-146a and correlates with an increased risk to several cancers (Jazdzewski et al., 2008; Hezova et al., 2012; Hung et al., 2012; Lung et al., 2012; Wang et al., 2012; Yamashita et al., 2013). This SNP has been shown to occur in the pre-miR-146a sequence and does not alter the sequence of mature miR-146a (Jazdzewski et al., 2008; Hezova et al., 2012; Hung et al., 2012; Lung et al., 2012; Wang et al., 2012; Yamashita et al., 2013). The mechanism by which this SNP promotes tumorigenesis and its potential role in melanomagenesis remain to be determined. To address this question, we expressed both pre-miR-146a/C and pre-miR-146a/G (Figure 3A) in highly-tumorigenic human melanoma cell lines that efficiently formed colonies in soft-agar and tumors in immunocompromised mice. Significantly, consistent with a previous report (Jazdzewski et al., 2008), the amount of mature miR-146a produced from pre-miR-146a/G was higher than that from pre-miR-146a/C (Figure 3—figure supplement 1). Ectopic expression of pre-miR-146a/G promoted proliferation at a higher rate than pre-miR-146a/C, as evidenced by increased colony formation and increased proliferation in two of the three melanoma cell lines analyzed (Figure 3B, Figure 3—figure supplement 2). We also compared the ability of pre-miR-146a/C and pre-miR-146a/G to promote anchorage-independent growth in soft-agar. Again, pre-miR-146a/G-stimulated colony formation more efficiently than pre-miR-146a/C (Figure 3C). Notably, although expression of pre-miR-146a/G in A375 cells did not increase proliferation in liquid culture (Figure 3B), it did increase colony formation in soft-agar (Figure 3C). Conversely, inhibition of miR-146a by miRZip-146a in SKMEL-28 and M14 cells reduced colony formation in liquid culture and soft-agar, and inhibited tumor formation in mice (Figure 3D–G and Figure 3—figure supplement 3). Similarly, expression of a miR-146a locked nucleic acid (LNA)-based antagomiR in SKMEL-28 and M14 cells reduced colony formation in liquid culture and soft-agar (Figure 3—figure supplements 4 and 5). By contrast, expression of a miR-146a antagomiR in YUSIV cells, which express low levels of miR-146a, did not significantly affect colony formation in either liquid culture or soft-agar (Figure 3—figure supplement 6).10.7554/eLife.01460.014Figure 3.Oncogenic activity of pre-miR-146a/C and pre-miR-146a/G.


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)

pre-miR-146a/G promotes proliferation of melanoma cells more effective than pre-miR-146a/C.M14, SKMEL-28 and A375 cells stably expressing pre-miR-146a/C (blue) or pre-miR-146a/G (red) or an empty vector (black) were analyzed for proliferation at indicated days. Relative proliferation is plotted. *, ** and *** represents p values <0.01, <0.001 and <0.0001 respectively.DOI:http://dx.doi.org/10.7554/eLife.01460.016
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3s2: pre-miR-146a/G promotes proliferation of melanoma cells more effective than pre-miR-146a/C.M14, SKMEL-28 and A375 cells stably expressing pre-miR-146a/C (blue) or pre-miR-146a/G (red) or an empty vector (black) were analyzed for proliferation at indicated days. Relative proliferation is plotted. *, ** and *** represents p values <0.01, <0.001 and <0.0001 respectively.DOI:http://dx.doi.org/10.7554/eLife.01460.016
Mentions: Several recent studies have found that a pre-miR-146a SNP (C>G rs2910164) alters the expression of mature miR-146a and correlates with an increased risk to several cancers (Jazdzewski et al., 2008; Hezova et al., 2012; Hung et al., 2012; Lung et al., 2012; Wang et al., 2012; Yamashita et al., 2013). This SNP has been shown to occur in the pre-miR-146a sequence and does not alter the sequence of mature miR-146a (Jazdzewski et al., 2008; Hezova et al., 2012; Hung et al., 2012; Lung et al., 2012; Wang et al., 2012; Yamashita et al., 2013). The mechanism by which this SNP promotes tumorigenesis and its potential role in melanomagenesis remain to be determined. To address this question, we expressed both pre-miR-146a/C and pre-miR-146a/G (Figure 3A) in highly-tumorigenic human melanoma cell lines that efficiently formed colonies in soft-agar and tumors in immunocompromised mice. Significantly, consistent with a previous report (Jazdzewski et al., 2008), the amount of mature miR-146a produced from pre-miR-146a/G was higher than that from pre-miR-146a/C (Figure 3—figure supplement 1). Ectopic expression of pre-miR-146a/G promoted proliferation at a higher rate than pre-miR-146a/C, as evidenced by increased colony formation and increased proliferation in two of the three melanoma cell lines analyzed (Figure 3B, Figure 3—figure supplement 2). We also compared the ability of pre-miR-146a/C and pre-miR-146a/G to promote anchorage-independent growth in soft-agar. Again, pre-miR-146a/G-stimulated colony formation more efficiently than pre-miR-146a/C (Figure 3C). Notably, although expression of pre-miR-146a/G in A375 cells did not increase proliferation in liquid culture (Figure 3B), it did increase colony formation in soft-agar (Figure 3C). Conversely, inhibition of miR-146a by miRZip-146a in SKMEL-28 and M14 cells reduced colony formation in liquid culture and soft-agar, and inhibited tumor formation in mice (Figure 3D–G and Figure 3—figure supplement 3). Similarly, expression of a miR-146a locked nucleic acid (LNA)-based antagomiR in SKMEL-28 and M14 cells reduced colony formation in liquid culture and soft-agar (Figure 3—figure supplements 4 and 5). By contrast, expression of a miR-146a antagomiR in YUSIV cells, which express low levels of miR-146a, did not significantly affect colony formation in either liquid culture or soft-agar (Figure 3—figure supplement 6).10.7554/eLife.01460.014Figure 3.Oncogenic activity of pre-miR-146a/C and pre-miR-146a/G.

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