DGCR8-mediated disruption of miRNA biogenesis induces cellular senescence in primary fibroblasts
Daniel Gómez-Cabello
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
These authors contributed equally.Search for more papers by this authorIsabel Adrados
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
These authors contributed equally.Search for more papers by this authorDavid Gamarra
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
Search for more papers by this authorHikaru Kobayashi
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
Search for more papers by this authorYoshihiro Takatsu
Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, London, UK
Search for more papers by this authorKyoko Takatsu
Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, London, UK
Search for more papers by this authorJesús Gil
Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, London, UK
Search for more papers by this authorCorresponding Author
Ignacio Palmero
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
Correspondence
Ignacio Palmero, Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM. Arturo Duperier, 4, Madrid 28029, Spain. Tel.: (+34) 915854491; fax: (+34) 915854401; e-mail: [email protected]
Search for more papers by this authorDaniel Gómez-Cabello
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
These authors contributed equally.Search for more papers by this authorIsabel Adrados
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
These authors contributed equally.Search for more papers by this authorDavid Gamarra
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
Search for more papers by this authorHikaru Kobayashi
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
Search for more papers by this authorYoshihiro Takatsu
Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, London, UK
Search for more papers by this authorKyoko Takatsu
Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, London, UK
Search for more papers by this authorJesús Gil
Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, London, UK
Search for more papers by this authorCorresponding Author
Ignacio Palmero
Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM, Madrid, Spain
Correspondence
Ignacio Palmero, Instituto de Investigaciones Biomédicas ‘Alberto Sols’ CSIC-UAM. Arturo Duperier, 4, Madrid 28029, Spain. Tel.: (+34) 915854491; fax: (+34) 915854401; e-mail: [email protected]
Search for more papers by this authorSummary
The regulation of gene expression by microRNAs (miRNAs) is critical for normal development and physiology. Conversely, miRNA function is frequently impaired in cancer, and other pathologies, either by aberrant expression of individual miRNAs or dysregulation of miRNA synthesis. Here, we have investigated the impact of global disruption of miRNA biogenesis in primary fibroblasts of human or murine origin, through the knockdown of DGCR8, an essential mediator of the synthesis of canonical miRNAs. We find that the inactivation of DGCR8 in these cells results in a dramatic antiproliferative response, with the acquisition of a senescent phenotype. Senescence triggered by DGCR8 loss is accompanied by the upregulation of the cell-cycle inhibitor p21CIP1. We further show that a subset of senescence-associated miRNAs with the potential to target p21CIP1 is downregulated during DGCR8-mediated senescence. Interestingly, the antiproliferative response to miRNA biogenesis disruption is retained in human tumor cells, irrespective of p53 status. In summary, our results show that defective synthesis of canonical microRNAs results in cell-cycle arrest and cellular senescence in primary fibroblasts mediated by specific miRNAs, and thus identify global miRNA disruption as a novel senescence trigger.
Supporting Information
| Filename | Description |
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| acel12117-sup-0001-FigureS1-S6.pdfapplication/PDF, 4 MB | Fig. S1 Antiproliferative effect of abrogation of DGCR8 in fibroblasts. (A) QPCR analysis of the silencing efficiency of the indicated shRNA vectors, transcript levels for Dicer, Drosha or DGCR8 was normalized to the levels in empty vector-infected cells. (B) Long term proliferation of IMR-90 human fibroblasts retrovirally infected with two shRNAs against DGCR8. (C) Crystal violet staining of IMR-90 human fibroblasts retrovirally infected as in A. (D) Growth curve of IMR-90 human fibroblasts infected with two independent shRNAs against Dicer or Drosha, or an empty vector. Fig. S2. (A) Western blot analysis of the indicated proteins in IMR90 fibroblasts, after infection with shRNA vectors. (B) Western blot analysis of the indicated proteins in MEF of the indicated genotypes, after infection with shRNA vectors. Erk, total Erk1/2 protein; P-Erk, phosphorylated Erk1/2. Fig. S3 (A) Representative images of DAPI staining of nuclei from MEFs of the indicated genotype, top, or IMR-90 human fibroblasts, bottom, expressing the indicated shRNA vectors. (B) Percentage of nuclei positive for gamma-H2AX staining in IMR-90 and BJ fibroblasts. BJ fibroblasts irradiated with 10 Gy of gamma irradiation are shown as a positive control. Fig. S4 Schematic representation of target sequences for the indicated miRNAs in the 3′UTR region of human p21CIP1, showing their complementarity to seed sequences of the miRNAs. Fig. S5 (A) Relative cell number estimated by crystal violet staining of HCT-116 and HCT-116 p53KO cells after silencing of DGCR8. (B) Western blot analysis of the indicated proteins in U2OS or 293T cells, after infection and selection (U2OS) or transient transfection (293T) with the indicated shRNA vectors. Fig. S6 Cell-cycle regulators with differential expression in shDGCR8 IMR90 cells. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- Abad M, Moreno A, Palacios A, Narita M, Blanco F, Moreno-Bueno G, Palmero I (2011) The tumor suppressor ING1 contributes to epigenetic control of cellular senescence. Aging Cell 10, 158–171.
- Bonifacio LN, Jarstfer MB (2010) miRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts. PLoS ONE 5, e12519.
- Borgdorff V, Lleonart ME, Bishop CL, Fessart D, Bergin AH, Overhoff MG, Beach DH (2010) Multiple microRNAs rescue from Ras-induced senescence by inhibiting p21(Waf1/Cip1). Oncogene 29, 2262–2271.
- Brown JP, Wei W, Sedivy JM (1997) Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 277, 831–834.
- Chen Z, Wu J, Yang C, Fan P, Balazs L, Jiao Y, Lu M, Gu W, Li C, Pfeffer LM, Tigyi G, Yue J (2012) DiGeorge syndrome critical region 8(DGCR8) -mediated miRNA biogenesis is essential for vascular smooth muscle cell development in mice. J. Biol. Chem. 287, 19018–19028.
- Collado M, Serrano M (2010) Senescence in tumours: evidence from mice and humans. Nat. Rev. Cancer 10, 51–57.
- Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C, Schurra C, Garre M, Nuciforo PG, Bensimon A, Maestro R, Pelicci PG, d'Adda di Fagagna F (2006) Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 444, 638–642.
- Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, Peacocket M, Campisi J. (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. U.S.A. 92, 9363–9367.
- Esteller M (2011) Non-coding RNAs in human disease. Nat. Rev. Genet. 12, 861–874.
- Faraonio R, Salerno P, Passaro F, Sedia C, Iaccio A, Bellelli R, Nappi TC, Comegna M, Romano S, Salvatore G, Santoro M, Cimino F (2012) A set of miRNAs participates in the cellular senescence program in human diploid fibroblasts. Cell Death Differ. 19, 713–721.
- Fenelon K, Mukai J, Xu B, Hsu PK, Drew LJ, Karayiorgou M, Fischbach GD, Macdermott AB, Gogos JA (2011) Deficiency of Dgcr8, a gene disrupted by the 22q11.2 microdeletion, results in altered short-term plasticity in the prefrontal cortex. Proc. Natl Acad. Sci. U.S.A. 108, 4447–4452.
- Francia S, Michelini F, Saxena A, Tang D, de Hoon M, Anelli V, Mione M, Carninci P, d'Adda di Fagagna F (2012) Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488, 231–235.
- Gomez-Cabello D, Callejas S, Benguria A, Moreno A, Alonso J, Palmero I (2010) Regulation of the microRNA processor DGCR8 by the tumor suppressor ING1. Cancer Res. 70, 1866–1874.
- Gorospe M, Abdelmohsen K (2011) MicroRegulators come of age in senescence. Trends Genet. 27, 233–241.
- Greussing R, Hackl M, Charoentong P, Pauck A, Monteforte R, Cavinato M, Hofer E, Scheideler M, Neuhaus M, Micutkova L, Mueck C, Trajanoski Z, Grillari J, Jansen-Dürr P (2013) Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts. BMC Genomics 14, 224.
- Guo H, Ingolia NT, Weissman JS, Bartel DP (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840.
- Ivanovska I, Ball AS, Diaz RL, Magnus JF, Kibukawa M, Schelter JM, Kobayashi SV, Lim L, Burchard J, Jackson AL, Linsley PS, Cleary MA (2008) MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression. Mol. Cell. Biol. 28, 2167–2174.
- Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D, Slack FJ (2005) RAS is regulated by the let-7 microRNA family. Cell 120, 635–647.
- Kuilman T, Michaloglou C, Mooi WJ, Peeper DS (2010) The essence of senescence. Genes Dev. 24, 2463–2479.
- Kumar MS, Lu J, Mercer KL, Golub TR, Jacks T (2007) Impaired microRNA processing enhances cellular transformation and tumorigenesis. Nat. Genet. 39, 673–677.
- Liang XH, Crooke ST (2011) Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing. Nucleic Acids Res. 39, 4875–4889.
- Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR (2005) MicroRNA expression profiles classify human cancers. Nature 435, 834–838.
- Lujambio A, Lowe SW (2012) The microcosmos of cancer. Nature 482, 347–355.
- Marasa BS, Srikantan S, Martindale JL, Kim MM, Lee EK, Gorospe M, Abdelmohsen K (2010) MicroRNA profiling in human diploid fibroblasts uncovers miR-519 role in replicative senescence. Aging 2, 333–343.
- Melo SA, Moutinho C, Ropero S, Calin GA, Rossi S, Spizzo R, Fernandez AF, Davalos V, Villanueva A, Montoya G, Yamamoto H, Schwartz S Jr, Esteller M (2010) A genetic defect in exportin-5 traps precursor microRNAs in the nucleus of cancer cells. Cancer Cell 18, 303–315.
- Melton C, Blelloch R (2010) MicroRNA Regulation of Embryonic Stem Cell Self-Renewal and Differentiation. Adv. Exp. Med. Biol. 695, 105–117.
- Mudhasani R, Zhu Z, Hutvagner G, Eischen CM, Lyle S, Hall LL, Lawrence JB, Imbalzano AN, Jones SN (2008) Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells. J. Cell Biol. 181, 1055–1063.
- Muralidhar B, Goldstein LD, Ng G, Winder DM, Palmer RD, Gooding EL, Barbosa-Morais NL, Mukherjee G, Thorne NP, Roberts I, Pett MR, Coleman N (2007) Global microRNA profiles in cervical squamous cell carcinoma depend on Drosha expression levels. J. Pathol. 212, 368–377.
- Palmero I, Pantoja C, Serrano M (1998) p19ARF links the tumour suppressor p53 to Ras. Nature 395, 125–126.
- Palmero I, Murga M, Zubiaga A, Serrano M (2002) Activation of ARF by oncogenic stress in mouse fibroblasts is independent of E2F1 and E2F2. Oncogene 21, 2939–2947.
- Pantoja C, Serrano M (1999) Murine fibroblasts lacking p21 undergo senescence and are resistant to transformation by oncogenic Ras. Oncogene 18, 4974–4982.
- Peric D, Chvalova K, Rousselet G (2012) Identification of microprocessor-dependent cancer cells allows screening for growth-sustaining micro-RNAs. Oncogene 31, 2039–2048.
- Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW (1997) Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593–602.
- Srikantan S, Gorospe M, Abdelmohsen K (2011) Senescence-associated microRNAs linked to tumorigenesis. Cell Cycle 10, 3211–3212.
- Stark KL, Xu B, Bagchi A, Lai WS, Liu H, Hsu R, Wan X, Pavlidis P, Mills AA, Karayiorgou M, Gogos JA (2008) Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model. Nat. Genet. 40, 751–760.
- Sugito N, Ishiguro H, Kuwabara Y, Kimura M, Mitsui A, Kurehara H, Ando T, Mori R, Takashima N, Ogawa R, Fujii Y (2006) RNASEN regulates cell proliferation and affects survival in esophageal cancer patients. Clin. Cancer Res. 12, 7322–7328.
- Thomson JM, Newman M, Parker JS, Morin-Kensicki EM, Wright T, Hammond SM (2006) Extensive post-transcriptional regulation of microRNAs and its implications for cancer. Genes Dev. 20, 2202–2207.
- Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, van Duijse J, Drost J, Griekspoor A, Zlotorynski E, Yabuta N, De Vita G, Nojima H, Looijenga LH, Agami R (2006) A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124, 1169–1181.
- Wang Y, Medvid R, Melton C, Jaenisch R, Blelloch R (2007) DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat. Genet. 39, 380–385.
- Wang Y, Baskerville S, Shenoy A, Babiarz JE, Baehner L, Blelloch R (2008) Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat. Genet. 40, 1478–1483.
- Wang M, Cheng Z, Tian T, Chen J, Dou F, Guo M, Cong YS (2011) Differential expression of oncogenic miRNAs in proliferating and senescent human fibroblasts. Mol. Cell. Biochem. 352, 271–279.
- Winter J, Jung S, Keller S, Gregory RI, Diederichs S (2009) Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat. Cell Biol. 11, 228–234.
- Wu S, Huang S, Ding J, Zhao Y, Liang L, Liu T, Zhan R, He X (2010) Multiple microRNAs modulate p21Cip1/Waf1 expression by directly targeting its 3′ untranslated region. Oncogene 29, 2302–2308.
- Yi R, Pasolli HA, Landthaler M, Hafner M, Ojo T, Sheridan R, Sander C, O'Carroll D, Stoffel M, Tuschl T, Fuchs E (2009) DGCR8-dependent microRNA biogenesis is essential for skin development. Proc. Natl Acad. Sci. U.S.A. 106, 498–502.
- Zindy F, Quelle DE, Roussel MF, Sherr CJ (1997) Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging. Oncogene 15, 203–211.
