Abstract
Although recent studies have shown that adenosine-to-inosine (A-to-I) RNA editing occurs in microRNAs (miRNAs), its effects on tumour growth and metastasis are not well understood. We present evidence of CREB-mediated low expression of ADAR1 in metastatic melanoma cell lines and tumour specimens. Re-expression of ADAR1 resulted in the suppression of melanoma growth and metastasis in vivo. Consequently, we identified three miRNAs undergoing A-to-I editing in the weakly metastatic melanoma but not in strongly metastatic cell lines. One of these miRNAs, miR-455-5p, has two A-to-I RNA-editing sites. The biological function of edited miR-455-5p is different from that of the unedited form, as it recognizes a different set of genes. Indeed, wild-type miR-455-5p promotes melanoma metastasis through inhibition of the tumour suppressor gene CPEB1. Moreover, wild-type miR-455 enhances melanoma growth and metastasis in vivo, whereas the edited form inhibits these features. These results demonstrate a previously unrecognized role for RNA editing in melanoma progression.
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Acknowledgements
We thank S. Maas from NIH for providing the ADAR1 A-to-I RNA-editing hairpin loop luciferase reporter plasmid. We thank W. Choi for her help with the miRNA microarray. We thank R. Rajesha for his technical help and support. These studies are supported by SINF, an MDACC grant and NIH Skin Cancer SPORE p50 CA093459.
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M.B-E. conceived and supervised the project. E.S. and A.K.M. designed and carried out experiments and analysed most of the data. R.R.B., T.K., L.H., M.E.V., G.V-T. and A.S. carried out experiments and analysed data. H.J.L., S.J.K. and I.J.F. helped with the spontaneous animal model. C.I., A.G.R., K.M.N., K.Z., D.B., S.J.M.J., I.B., M.M., Y-y.W., A.K.E., P.H. and J.E.G. carried out sequencing and analysis. A.K.S., G.A.C. and G.M. helped with miRNA analysis.
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Integrated supplementary information
Supplementary Figure 1 ADAR2 is not regulated by CREB.
Western blot analyses demonstrating that overexpression of CREB in SB2 cells and silencing CREB in C8161 cells did not affect the expression of ADAR2 in these cells. Note a doublet bands in ADAR2 expression in SB2 cells which represent two different isoforms (73,76KDa) (data are representative of 3, biologically independent experiments). Uncropped images of the blots are shown in Supplementary Figure 8.
Supplementary Figure 2 Luciferase imaging of lymph node metastasis in nude mice.
Images of mice after the primary tumors were removed show the presence or absence of local lymph node metastases. These mice represent the results described in Figure 4C (n = 8 mice in each group).
Supplementary Figure 3 Identification of miRs in common between CREB shRNA and ADAR1 shRNA arrays.
(A) Venn diagram showing the breakdown of the differentially regulated miRNAs between the two arrays analyzed. (B) Table of the 12 miRNAs identified to be in common between both the CREB shRNA and the ADAR1 shRNA array.
Supplementary Figure 4 miR-378-3p and miR-324-5p are edited by ADAR1 at one A-to-I editing site.
(A) Mature miR-378 sequence highlighted in green. The arrow indicates the RNA editing site. (B) DNA sequencing data indicates a decrease in A-to-I editing in SB2 cells when ADAR1 is silenced. (C) An increase in RNA editing in C8161 cells is observed when ADAR1 is overexpressed. (D) A-to-I RNA editing is increased on CREB silencing and is returned to NT levels on rescue of CREB in C8161 cells. Editing sites are indicated by a red arrow. (E) Mature miR-324 sequence is highlighted in green. The arrow indicates the RNA editing site. (F) DNA sequencing data indicates a decrease in A-to-I editing in SB2 cells when ADAR1 is silenced. (G) An increase in RNA editing is observed in C8161 cells when ADAR1 is overexpressed. (H) A-to-I RNA editing is increased on CREB silencing and is returned to NT levels on rescue of CREB in C8161 cells. Editing sites are indicated by a red arrow.
Supplementary Figure 5 Bound miR-455 to Drosha and Dicer correlates with ADAR1 expression.
(A) Silencing ADAR1 in SB2 melanoma cells results in increased binding of mir-455 to Dicer, (B) Overexpressing ADAR1 in C8161 melanoma cells reduced the binding of mir-455 to Dicer. (C). Overexpression of ADAR1 in C8161 melanoma cells reduced the amount of pri-miR-455 bound to Drosha. (D) Silencing ADAR1 in SB2 melanoma cells resulted with increased interaction between pri-miR-455 and Drosha (n = 3 biologically independent samples; statistical significance by two-tailed Student t-test; error bars represent s.d., p < 0.05).
Supplementary Figure 6 Confirmation of manipulation of miR-455-5p levels.
(A) rtPCR analysis of miR-455-5p levels show that wild-type miR-455-5p is overexpressed 2 fold, edited1 miR-455-5p is overexpressed ∼20 fold, edited2 miR-455-5p is overexpressed ∼9 fold and double edited miR-455-5p is overexpressed ∼40 fold, as compared to empty vector control. The antagomir to miR-455-5p shows a downregulation of miR-455-5p by about 90% as compared to the NT control (n = 3 biologically independent samples; statistical significance was determined by two-tailed Student t-test; error bars represent s.d., p < 0.05). (B) Rescue of CPEB1 expression in SB2 ADAR1 KD cells inhibits their invasive potential in matrigel invasion assay, without affecting their proliferation rate (n = 3 biologically independent samples; statistical significance was determined by Tukey’s multiple comparison test; error bars represent s.d., p < 0.05). (C) No change was observed in proliferation rate between SB2 cells transfected with ADAR1-NT, ADAR1-KD, and ADAR1-KD CPEB1-OE (n = 12 biologically independent samples per goup. Statistical significance was determined by one-way ANOVA; error bars represent s.d., p = 0.9610).
Supplementary Figure 7 Potential edited miR-455-5p binding sites on 3'UTR of ITGα2, MDM4 and RhoC.
miR-455-5p seed sequence is depicted in red and the edited sites are shown in green. 6-mer or 7-mer binding sites are highlighted in yellow.
Supplementary Figure 8
Uncropped Western blots and gel images.
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Shoshan, E., Mobley, A., Braeuer, R. et al. Reduced adenosine-to-inosine miR-455-5p editing promotes melanoma growth and metastasis. Nat Cell Biol 17, 311–321 (2015). https://doi.org/10.1038/ncb3110
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DOI: https://doi.org/10.1038/ncb3110
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