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Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma

Abstract

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.

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Figure 1: The LIN28B/let-7/MYCN axis is intact in neuroblastoma.
Figure 2: LIN28B is dispensable in human MYCN amplified neuroblastoma cells.
Figure 3: MYCN mRNA is a ceRNA for let-7.
Figure 4: Loss of let-7a2 and let-7g is common in neuroblastoma.
Figure 5: The MYCN aceRNA model predicts let-7 chromosomal loss patterns in neuroblastoma.

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References

  1. Croce, C. M. Causes and consequences of microRNA dysregulation in cancer. Nature Rev. Genet. 10, 704–714 (2009)

    CAS  PubMed  Google Scholar 

  2. Johnson, S. M. et al. RAS is regulated by the let-7 microRNA family. Cell 120, 635–647 (2005)

    CAS  PubMed  Google Scholar 

  3. Sampson, V. B. et al. MicroRNA let-7a down-regulates MYC and reverts MYC-induced growth in Burkitt lymphoma cells. Cancer Res. 67, 9762–9770 (2007)

    CAS  PubMed  Google Scholar 

  4. Mayr, C., Hemann, M. T. & Bartel, D. P. Disrupting the pairing between let-7 and Hmga2 enhances oncogenic transformation. Science 315, 1576–1579 (2007)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–838 (2005)

    ADS  CAS  PubMed  Google Scholar 

  6. Boyerinas, B., Park, S.-M., Hau, A., Murmann, A. E. & Peter, M. E. The role of let-7 in cell differentiation and cancer. Endocr. Relat. Cancer 17, F19–F36 (2010)

    CAS  PubMed  Google Scholar 

  7. Gurtan, A. M. & Sharp, P. A. The role of miRNAs in regulating gene expression networks. J. Mol. Biol. 425, 3582–3600 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Blandino, G. et al. Tumor suppressor microRNAs: a novel non-coding alliance against cancer. FEBS Lett. 588, 2639–2652 (2014)

    CAS  PubMed  Google Scholar 

  9. Viswanathan, S. R., Daley, G. Q. & Gregory, R. I. Selective blockade of microRNA processing by Lin28. Science 320, 97–100 (2008)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Nguyen, L. H. et al. Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models. Cancer Cell 26, 248–261 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Viswanathan, S. R. et al. Lin28 promotes transformation and is associated with advanced human malignancies. Nature Genet. 41, 843–848 (2009)

    CAS  PubMed  Google Scholar 

  12. Molenaar, J. J. et al. LIN28B induces neuroblastoma and enhances MYCN levels via let-7 suppression. Nature Genet. 44, 1199–1206 (2012)

    CAS  PubMed  Google Scholar 

  13. Diskin, S. J. et al. Common variation at 6q16 within HACE1 and LIN28B influences susceptibility to neuroblastoma. Nature Genet. 44, 1126–1130 (2012)

    CAS  PubMed  Google Scholar 

  14. Madison, B. B. et al. LIN28B promotes growth and tumorigenesis of the intestinal epithelium via Let-7. Genes Dev. 27, 2233–2245 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Urbach, A. et al. Lin28 sustains early renal progenitors and induces Wilms tumor. Genes Dev. 28, 971–982 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Tu, H. C. et al. LIN28 cooperates with WNT signaling to drive invasive intestinal and colorectal adenocarcinoma in mice and humans. Genes Dev. 29, 1074–1086 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Tay, Y., Rinn, J. & Pandolfi, P. P. The multilayered complexity of ceRNA crosstalk and competition. Nature 505, 344–352 (2014)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  18. Poliseno, L. et al. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465, 1033–1038 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cesana, M. & Daley, G. Q. Deciphering the rules of ceRNA networks. Proc. Natl Acad. Sci. USA 110, 7112–7113 (2013)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005)

    CAS  PubMed  Google Scholar 

  21. Melton, C., Judson, R. L. & Blelloch, R. Opposing microRNA families regulate self-renewal in mouse embryonic stem cells. Nature 463, 621–626 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Baeyens, K. J., De Bondt, H. L., Pardi, A. & Holbrook, S. R. A curved RNA helix incorporating an internal loop with G·A and A·A non-Watson–Crick base pairing. Proc. Natl Acad. Sci. USA 93, 12851–12855 (1996)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  23. Pugh, T. J. et al. The genetic landscape of high-risk neuroblastoma. Nature Genet. 45, 279–284 (2013)

    CAS  PubMed  Google Scholar 

  24. Molenaar, J. J. et al. Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes. Nature 483, 589–593 (2012)

    ADS  CAS  PubMed  Google Scholar 

  25. Barone, G., Anderson, J., Pearson, A. D. J., Petrie, K. & Chesler, L. New strategies in neuroblastoma: therapeutic targeting of MYCN and ALK. Clin. Cancer Res. 19, 5814–5421 (2013)

    CAS  PubMed  Google Scholar 

  26. Maris, J. M. Recent advances in neuroblastoma. N. Engl. J. Med. 362, 2202–2211 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Seeger, R. C. et al. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N. Engl. J. Med. 313, 1111–1116 (1985)

    CAS  PubMed  Google Scholar 

  28. Brodeur, G. M., Seeger, R. C., Schwab, M., Varmus, H. E. & Bishop, J. M. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 224, 1121–1124 (1984)

    ADS  CAS  PubMed  Google Scholar 

  29. Ala, U. et al. Integrated transcriptional and competitive endogenous RNA networks are cross-regulated in permissive molecular environments. Proc. Natl Acad. Sci. USA 110, 7154–7159 (2013)

    ADS  MathSciNet  CAS  PubMed  PubMed Central  Google Scholar 

  30. Denzler, R., Agarwal, V., Stefano, J., Bartel, D. P. & Stoffel, M. Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance. Mol. Cell 54, 766–776 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Bosson, A. D., Zamudio, J. R. & Sharp, P. A. Endogenous miRNA and target concentrations determine susceptibility to potential ceRNA competition. Mol. Cell 56, 347–359 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Agarwal, V., Bell, G. W., Nam, J.-W. & Bartel, D. P. Predicting effective microRNA target sites in mammalian mRNAs. eLife 4, http://dx.doi.org/10.7554/eLife.05005 (2015)

  33. Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Beckers, A. et al. MYCN-targeting miRNAs are predominantly downregulated during MYCN-driven neuroblastoma tumor formation. Oncotarget 6, 5204–5216 (2015)

    PubMed  Google Scholar 

  35. Maris, J. M. et al. Allelic deletion at chromosome bands 11q14-23 is common in neuroblastoma. Med. Pediatr. Oncol. 36, 24–27 (2001)

    CAS  PubMed  Google Scholar 

  36. Breen, C. J., O’Meara, A., McDermott, M., Mullarkey, M. & Stallings, R. L. Coordinate deletion of chromosome 3p and 11q in neuroblastoma detected by comparative genomic hybridization. Cancer Genet. Cytogenet. 120, 44–49 (2000)

    CAS  PubMed  Google Scholar 

  37. Ejeskär, K., Aburatani, H., Abrahamsson, J., Kogner, P. & Martinsson, T. Loss of heterozygosity of 3p markers in neuroblastoma tumours implicate a tumour-suppressor locus distal to the FHIT gene. Br. J. Cancer 77, 1787–1791 (1998)

    PubMed  Google Scholar 

  38. Bray, I. et al. Widespread dysregulation of MiRNAs by MYCN amplification and chromosomal imbalances in neuroblastoma: association of miRNA expression with survival. PLoS ONE 4, e7850 (2009)

    ADS  PubMed  PubMed Central  Google Scholar 

  39. Chiang, H. R. et al. Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. Genes Dev. 24, 992–1009 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Roush, S. & Slack, F. J. The let-7 family of microRNAs. Trends Cell Biol. 18, 505–516 (2008)

    CAS  PubMed  Google Scholar 

  41. Hackett, C. S. et al. Genome-wide array CGH analysis of murine neuroblastoma reveals distinct genomic aberrations which parallel those in human tumors. Cancer Res. 63, 5266–5273 (2003)

    CAS  PubMed  Google Scholar 

  42. Karreth, F. A. et al. The BRAF pseudogene functions as a competitive endogenous RNA and induces lymphoma in vivo. Cell 161, 319–332 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Maris, J. M., Hogarty, M. D., Bagatell, R. & Cohn, S. L. Neuroblastoma. Lancet 369, 2106–2120 (2007)

    CAS  PubMed  Google Scholar 

  44. Pugh, T. J. et al. Exome sequencing of pleuropulmonary blastoma reveals frequent biallelic loss of TP53 and two hits in DICER1 resulting in retention of 5p-derived miRNA hairpin loop sequences. Oncogene 33, 5295–5302 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Rakheja, D. et al. Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours. Nature Commun. 2, 4802 (2014)

    CAS  Google Scholar 

  46. Iwakawa, R. et al. Genome-wide identification of genes with amplification and/or fusion in small cell lung cancer. Genes Chromosom. Cancer 52, 802–816 (2013)

    CAS  PubMed  Google Scholar 

  47. Thériault, B. L., Dimaras, H., Gallie, B. L. & Corson, T. W. The genomic landscape of retinoblastoma: a review. Clin. Experiment. Ophthalmol. 42, 33–52 (2014)

    PubMed  Google Scholar 

  48. Mayr, C. & Bartel, D. P. Widespread shortening of 3′ UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138, 673–684 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Iwasaki, S., Kawamata, T. & Tomari, Y. Drosophila argonaute1 and argonaute2 employ distinct mechanisms for translational repression. Mol. Cell 34, 58–67 (2009)

    CAS  PubMed  Google Scholar 

  50. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nature Methods 12, 357–360 (2015)

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Hafner, M. et al. RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA 17, 1697–1712 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Patro, R., Duggal, G. & Kingsford, C. Salmon: accurate, versatile and ultrafast quantification from RNA-seq data using lightweight-alignment. bioRxiv http://dx.doi.org/10.1101/021592 (2015)

  53. Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nature Methods 11, 783–784 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014)

    ADS  CAS  PubMed  Google Scholar 

  55. Spitz, R. et al. Oligonucleotide array-based comparative genomic hybridization (aCGH) of 90 neuroblastomas reveals aberration patterns closely associated with relapse pattern and outcome. Genes Chromosom. Cancer 45, 1130–1142 (2006)

    CAS  PubMed  Google Scholar 

  56. Thiessen, J. et al. Chromosome 17/17q gain and unaltered profiles in high resolution array-CGH are prognostically informative in neuroblastoma. Genes Chromosom. Cancer 53, 639–649 (2014)

    Google Scholar 

  57. Rushlow, D. E. et al. Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies. Lancet Oncol. 14, 327–334 (2013)

    CAS  PubMed  Google Scholar 

  58. Kent, W. J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

G.Q.D. is supported by National Institutes of Health grant R01GM107536, Alex’s Lemonade Stand Foundation, and the Ellison Medical Foundation. G.Q.D. is an affiliate member of the Broad Institute, and an investigator of the Howard Hughes Medical Institute and the Manton Center for Orphan Disease Research. J.T.P. was supported by Alex’s Lemonade Stand Foundation. K.M.T. was supported as a Howard Hughes Medical Institute International Student Research Fellow and as a Herchel Smith Graduate Fellow. D.S.P. and R.E. were supported by award number T32GM007753 from the National Institute of General Medical Sciences.

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Authors and Affiliations

Authors

Contributions

G.Q.D. provided support and guidance for this work; G.Q.D. and J.T.P. conceived the hypothesis, designed the study, and wrote the manuscript; J.T.P. performed and interpreted most of the experiments and generated figures; K.M.T. helped perform the shRNA and siRNA experiments and generated most of the plasmid constructs; D.S.P. helped perform the RNA sequencing experiments; F.R., J.T., and F.B. generated the aCGH and survival data on neuroblastoma patients; C.S.S., K.C.K., and J.J.C. acquired tissue samples and assisted with the IHC analysis; R.E. assisted with the CRISPR experiments; M.S., Y.d.S., G.S.LP., and S.J.R. provided technical help; P.C. processed RNA sequencing data and helped with data analysis; H.C.T. helped perform in vitro transfection experiments; A.H. assisted with RNA sequencing data analysis; J.K.O. and C.S.S. performed xenograft experiments; M.C. assisted with the RNA sequencing experiments.

Corresponding author

Correspondence to George Q. Daley.

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Competing interests

G.Q.D. holds options and intellectual property relating to 28/7 Therapeutics, a company seeking to develop inhibitors of the LIN28/let-7 pathway.

Extended data figures and tables

Extended Data Figure 1 MYCN is a highly conserved let-7 target.

a, Schematic of human MYCN ORF and 3′ UTR, indicating let-7 sites 1 and 2 and their approximate location. b, Predicted base pairing patterns of let-7a, let-7f, and let-7g with MYCN let-7 sites 1 and 2. A–G base pairs, common in RNA, are represented by an asterisk. c, Alignments of let-7 sites 1 and 2 in 100 vertebrate MYCN 3′ UTRs (ENCODE, https://genome.ucsc.edu/ENCODE/).

Extended Data Figure 2 LIN28B expression and function in neuroblastoma.

a, MYCN, LIN28B, and LIN28A mRNA expression levels in neuroblastoma (n = 649; see Source Data (ED Fig 2) in Supplementary Information). b, Immunoblot for indicated proteins in human embryonic carcinoma cells (PA1), normal human fibroblasts (HF), SK-N-SH (SH), SK-N-AS (AS), SK-N-F1 (F1), BE2C (BE), SK-N-DZ (DZ), Kelly (Ke), and human chronic myeloid leukaemia cells (K5). For gel source data, see Supplementary Figures. c, Representative LIN28B immunohistochemical staining of human neuroblastoma by stage (left), percentage LIN28B positive neuroblastoma by disease stage (right); (n = 36). GNB, ganglioneuroblastoma. d, LIN28B expression by neuroblastoma stage (n = 64; Source Data (ED Fig 2)). e, Immunoblot for LIN28B in inducible LIN28B SH-SY5Y cells and GFP- or LIN28B-expressing SK-N-AS cells (left) and corresponding qPCR analysis of relative let-7 family levels (right) (mean plus s.e.m. of three independent experiments shown). f, Relative growth rate (BrdU incorporation, right) of SH-SY5Y and SK-N-AS neuroblastoma cells from d (*P < 0.05, n = 3 independent experiments).

Source data

Extended Data Figure 3 Short hairpin knockdown of LIN28B in neuroblastoma.

a, Immunoblot for indicated proteins MYCN and LIN28B in MYCN-amplified cells infected with LIN28B targeting lentiviral shRNAs. For gel source data, see Supplementary Figures. b, Cell proliferation analysis of cells described in a (n = 3 independent experiments). c, Average tumour size of human–mouse subcutaneous xenograft tumour analysis 3 weeks after injection of 2 × 106 cells infected with a LIN28B targeting lentiviral shRNA (n = 6 mice for BE(2)C, n = 3 mice for SK-N-DZ; Supplementary Figures and Source Data (ED Fig 3)). d, qPCR analysis of let-7a, let-7b, and let-7i levels in cells described in a (mean plus s.e.m. of three independent experiments shown). e, Cell proliferation analysis of BE(2)C cells stably expressing red fluorescence protein (RFP), Flag-tagged LIN28B ORF, or shRNA resistant Flag-tagged LIN28B (LIN28B shRes) infected with LIN28B lentiviral shRNAs targeting the LIN28B 3′ UTR (ShL28B-UTR) or the LIN28B open-reading frame (ShL28B-ORF). Cell counts were performed 7 days after lentiviral shRNA infection (mean plus s.e.m. of three independent experiments shown). f, Immunoblot for indicated proteins in cells described in e. For gel source data, see Supplementary Figures.

Source data

Extended Data Figure 4 Small interfering RNA knockdown of LIN28B in neuroblastoma.

a, Schematic of approximate siRNA target sites within the LIN28B mRNA. b, qPCR analysis of LIN28B mRNA levels in BE(2)C cells 48 h after transfection with the indicated LIN28B targeting siRNAs (mean of two independent experiments shown). c, Immunoblot analysis of LIN28B in cells from a. For gel source data, see Supplementary Figures. d, qPCR analysis of indicated let-7 levels in cells from a (mean of two independent experiments shown). e, Immunoblot analysis of MYCN and LIN28B in serially transfected MYCN-amplified cells for 6 or 9 days. Identical transfections were performed on days 0, 3, and 6. For gel source data, see Supplementary Figures. f, Day 9 qPCR analysis of the let-7 family in the cells from a (n = 3 independent experiments, mean plus s.e.m. shown). g, Cell growth analysis of day 0 to day 6 cells from a (BrdU incorporation, n = 3 independent experiments, mean plus s.e.m. shown). h, Lentiviral CRISPR-Cas9/LIN28B gRNA strategy targeting LIN28B at four distinct exon/intron junctions used in bg.

Extended Data Figure 5 Relative levels of let-7 targets in neuroblastoma.

a, mRNA-seq let-7 target table (as percentage let-7 target-site pool). b, qPCR analysis of indicated let-7 targets in neuroblastoma cells, PA1 embryonic carcinoma cells (EC), and normal human fibroblasts (hFib). Expression relative to β-ACTIN (ΔCT method) (mean of two biological replicates shown).

Extended Data Figure 6 Heat map of let-7 and small RNA spike reads.

Heat map of three BE(2)C and three Kelly sRNA-seq samples depicting the relative reads per million of the let-7 family, miR-17, and the six small RNA spikes added in equimolar amounts per sample (spikes miR-Neg, LET7A2, and LET7I were used to determine let-7 copies per cell from the small RNA sequencing data set). RPM, reads per million.

Extended Data Figure 7 qPCR quantification of MYCN and let-7 copies per cell.

a, Total let-7 sites per cell provided by MYCN mRNA in BE(2)C, Kelly, normal human fibroblasts (NHF), and embryonic carcinoma cells (EC) (mean plus s.e.m. of three biological replicates shown). b, Total let-7 copies per cell in cells from a, presented as stacked graphs of all let-7 family members (mean of three biological replicates shown). c, Total let-7 copies per cell in wild-type or LIN28B knockout BE(2)C and Kelly cells, presented as stacked graphs of all let-7 family members (values derived from let-7 copies per cell in b and average let-7 fold change described in Fig. 2f, g).

Extended Data Figure 8 Luciferase reporter and gain of function constructs.

a, Luciferase constructs used in the luciferase assays in Fig. 3d and Extended Data Fig. 8e. b, Schematic of the luciferase transfection protocols used in Fig. 3d. c, Schematic of the luciferase protocol used in Extended Data Fig. 8e. d, pcDNA3.1 constructs used in Extended Data Fig. 8e, f. e, Top: relative luciferase ratio in 293T cells co-transfected with the indicated 3′ UTR luciferase and pcDNA3.1 vectors in the presence of either control miRNA or let-7a mimic. Bottom: relative luciferase ratio in 293T cells co-transfected with the indicated 3′ UTR luciferase and pcDNA3.1 vectors in the presence of either a control miRNA or let-7a mimic. Mean of four independent experiments plus s.e.m. shown (*P < 0.05 relative to empty vector, unpaired t-test). f, Immunoblot analysis of MYCN in SK-N-AS cells stably expressing a MYCN ORF + 3′ UTR transgene and transfected with the indicated pcDNA3.1 vector. For gel source data, see Supplementary Figures.

Extended Data Figure 9 MYCN mRNA sponges let-7.

a, Immunoblot analysis of indicated proteins in BE(2)C cells transfected for 2.5 days with control, MYCN-1 (M1), or MYCN-2 (M2) siRNA and either control microRNA or let-7a inhibitor. For gel source data, see Supplementary Figures. b, qPCR analysis of DICER1, HK2, IMP1, LIN28B, and MYCN in cells transfected as in a. c, qPCR analysis of let-7a, let-7b, and let-7i in BE(2)C cells transfected for 2.5 days with control siRNA, siM1, or siM2 (n = 3 independent experiments, mean plus s.e.m. shown). d, Immunoblot analysis of indicated proteins in cells infected with indicated Cas9-gRNA lentivirus. For gel source data, see Supplementary Figures. e, Expression levels of let-7 targets in BE(2)C:MYCN cells transfected with siCon or siMYCN-3′ UTR. f, Relative let-7 expression in BE(2)C:MYCN cells co-transfected with siCon or siMYCN (3′ UTR) siRNA and miRCon or let-7a mimic. A 16-fold increase in let-7a results in an approximately eightfold increase in total let-7, owing to let-7a making up almost half of the total cellular pool (Fig. 3c, lower). g, Relative expression levels of let-7 targets in siCon and siMYCN cells transfected with let-7a mimic (data represent one round of mRNA-seq, ***P < 0.001, one-tailed Wilcoxon test, GSE81497, see Source Data F3).

Extended Data Figure 10 Neuroblastoma patient and ENCODE data.

a, Detail of the incidence of chromosome 3p21 and 11q23 loss and MYCN amplification as determined by analysis of the indicated retrospective chromosomal aberration studies on neuroblastoma. b, List of the ENCODE sRNA-seq samples analysed (with associated GEO accession numbers) for the relative expression of mature let-7 in Fig. 5c. c, List of let-7 family host transcripts, transcript class, and let-7 location within the transcript. d, List of the ENCODE mRNA-seq samples analysed (with associated University of California, Santa Cruz submission identifier numbers) for the relative expression of let-7 host transcripts in Fig. 5d. e, Relative expression of let-7a2, let-7f2, and let-7g host genes by microarray in MYCN-amplified and non-amplified neuroblastoma. ACTB shown as control. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired t-test, n = 643, Source Data (ED Fig 10). f, Schematic showing the several mechanisms that impair let-7 biogenesis and function in neuroblastoma (chromosome images created at http://www.ncbi.nlm.nih.gov/genome/tools/gdp/).

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Powers, J., Tsanov, K., Pearson, D. et al. Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma. Nature 535, 246–251 (2016). https://doi.org/10.1038/nature18632

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