Apoptosis

, Volume 18, Issue 7, pp 896–909 | Cite as

Ionizing radiation-inducible microRNA miR-193a-3p induces apoptosis by directly targeting Mcl-1

  • Jeong-Eun Kwon
  • Bu-Yeon Kim
  • Seo-Young Kwak
  • In-Hwa Bae
  • Young-Hoon Han
Original Paper

Abstract

The functions of microRNAs (miRNAs) as either oncogenes or tumor suppressors in regulating cancer-related events have been established. We analyzed the alterations in the miRNA expression profile of the glioma cell line U-251 caused by ionizing radiation (IR) by using an miRNA array and identified several miRNAs whose expression was significantly affected by IR. Among the IR-responsive miRNAs, we further examined the function of miR-193a-3p, which exhibited the most significant growth-inhibiting effect. miR-193a-3p was observed to induce apoptosis in both U-251 and HeLa cells. We also demonstrated that miR-193a-3p induces the accumulation of intracellular reactive oxygen species (ROS) and DNA damage as determined by the level of γH2AX and by performing the comet assay. The induction of both apoptosis and DNA damage by miR-193a-3p was blocked by antioxidant treatment, indicating the crucial role of ROS in the action of miR-193a-3p. Among the putative target proteins, the expression of Mcl-1, an anti-apoptotic Bcl-2 family member, decreased because of miR-193a-3p transfection. A reporter assay using a luciferase construct containing the 3′-untranslated region of Mcl-1 confirmed that Mcl-1 is a direct target of miR-193a-3p. Down-regulation of Mcl-1 by siRNA transfection closely mimicked the outcome of miR-193a-3p transfection showing increased ROS, DNA damage, cytochrome c release, and apoptosis. Ectopic expression of Mcl-1 suppressed the pro-apoptotic action of miR-193a-3p, suggesting that Mcl-1 depletion is critical for miR-193a-3p induced apoptosis. Collectively, our results suggest a novel function for miR-193a-3p and its potential application in cancer therapy.

Keywords

microRNA Apoptosis ROS miR-193a-3p Mcl-1 

References

  1. 1.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 16:281–297CrossRefGoogle Scholar
  2. 2.
    Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120:15–20PubMedCrossRefGoogle Scholar
  3. 3.
    Esquela-Kerscher A, Slack FJ (2006) Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 6:259–269PubMedCrossRefGoogle Scholar
  4. 4.
    Hwang HW, Mendell JT (2007) MicroRNAs in cell proliferation, cell death, and tumorigenesis. Br J Cancer 96(Suppl):R40–R44PubMedGoogle Scholar
  5. 5.
    Calin GA, Liu CG, Sevignani C, Ferracin M, Felli N, Dumitru CD, Shimizu M, Cimmino A, Zupo S, Dono M, Dell’Aquila ML, Alder H, Rassenti L, Kipps TJ, Bullrich F, Negrini M, Croce CM (2004) MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci U S A 101:11755–11760PubMedCrossRefGoogle Scholar
  6. 6.
    Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S, Shimizu M, Rattan S, Bullrich F, Negrini M, Croce CM (2004) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A 101:2999–3004PubMedCrossRefGoogle Scholar
  7. 7.
    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 435:834–838PubMedCrossRefGoogle Scholar
  8. 8.
    Cuconati A, Mukherjee C, Perez D, White E (2003) DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells. Genes Dev 17:2922–2932PubMedCrossRefGoogle Scholar
  9. 9.
    Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, Colman PM, Day CL, Adams JM, Huang DC (2005) Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 17:393–403PubMedCrossRefGoogle Scholar
  10. 10.
    Nijhawan D, Fang M, Traer E, Zhong Q, Gao W, Du F, Wang X (2003) Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. Genes Dev 17:1475–1486PubMedCrossRefGoogle Scholar
  11. 11.
    Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S, Deng X, Zhai D, Shi YX, Sneed T, Verhaegen M, Soengas M, Ruvolo VR, McQueen T, Schober WD, Watt JC, Jiffar T, Ling X, Marini FC, Harris D, Dietrich M, Estrov Z, McCubrey J, May WS, Reed JC, Andreeff M (2006) Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell 10:375–388PubMedCrossRefGoogle Scholar
  12. 12.
    Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD, Reed JC (1998) Elevated expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood 91:991–1000PubMedGoogle Scholar
  13. 13.
    Wuilleme-Toumi S, Robillard N, Gomez P, Moreau P, Le Gouill S, Avet-Loiseau H, Harousseau JL, Amiot M, Bataille R (2005) Mcl-1 is overexpressed in multiple myeloma and associated with relapse and shorter survival. Leukemia 19:1248–1252PubMedCrossRefGoogle Scholar
  14. 14.
    Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI, Adams JM, Huang DC (2005) Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes Dev 19:1294–1305PubMedCrossRefGoogle Scholar
  15. 15.
    Marsit CJ, Eddy K, Kelsey KT (2006) MicroRNA responses to cellular stress. Cancer Res 66:10843–10848PubMedCrossRefGoogle Scholar
  16. 16.
    Simone NL, Soule BP, Ly D, Saleh AD, Savage JE, Degraff W, Cook J, Harris CC, Gius D, Mitchell JB (2009) Ionizing radiation-induced oxidative stress alters miRNA expression. PLoS ONE 4:e6377PubMedCrossRefGoogle Scholar
  17. 17.
    Wagner-Ecker M, Schwager C, Wirkner U, Abdollahi A, Huber PE (2010) MicroRNA expression after ionizing radiation in human endothelial cells. Radiat Oncol 5:25PubMedCrossRefGoogle Scholar
  18. 18.
    Niemoeller OM, Niyazi M, Corradini S, Zehentmayr F, Li M, Lauber K, Belka C (2011) MicroRNA expression profiles in human cancer cells after ionizing radiation. Radiat Oncol 6:29PubMedCrossRefGoogle Scholar
  19. 19.
    Chistiakov DA, Chekhonin VP (2012) Contribution of microRNAs to radio- and chemoresistance of brain tumors and their therapeutic potential. Eur J Pharmacol 684:8–18PubMedCrossRefGoogle Scholar
  20. 20.
    Park SY, Lee JH, Ha M, Nam JW, Kim VN (2009) miR-29 miRNAs activate p53 by targeting p85 alpha and CDC42. Nat Struct Mol Biol 16:23–29PubMedCrossRefGoogle Scholar
  21. 21.
    Mott JL, Kobayashi S, Bronk SF, Gores GJ (2007) mir-29 regulates Mcl-1 protein expression and apoptosis. Oncogene 26:6133–6140PubMedCrossRefGoogle Scholar
  22. 22.
    Zhaohui P, David WV (1999) Analysis of redox regulation of cytochrome c-induced apoptosis in a cell-free system. Cell Death Differ 6:683–688CrossRefGoogle Scholar
  23. 23.
    Mills JR, Malina A, Pelletier J (2012) Inhibiting mitochondrial-dependent proteolysis of Mcl-1 promotes resistance to DNA damage. Cell Cycle 11:88–98PubMedCrossRefGoogle Scholar
  24. 24.
    Wensveen FM, Alves NL, Derks IA, Reedquist KA, Eldering E (2011) Apoptosis induced by overall metabolic stress converges on the Bcl-2 family proteins Noxa and Mcl-1. Apoptosis 16:708–721PubMedCrossRefGoogle Scholar
  25. 25.
    Ma K, He Y, Zhang H, Fei Q, Niu D, Wang D, Ding X, Xu H, Chen X, Zhu J (2012) DNA methylation-regulated miR-193a-3p dictates resistance of hepatocellular carcinoma to 5-fluorouracil via repression of SRSF2 expression. J Biol Chem 287:5639–5649PubMedCrossRefGoogle Scholar
  26. 26.
    Minagawa N, Kruglov EA, Dranoff JA, Robert ME, Gores GJ, Nathanson MH (2005) The anti-apoptotic protein Mcl-1 inhibits mitochondrial Ca2+ signals. J Biol Chem 280:33637–33644PubMedCrossRefGoogle Scholar
  27. 27.
    Warr MR, Mills JR, Nguyen M, Lemaire-Ewing S, Baardsnes J, Sun KL, Malina A, Young JC, Jeyaraju DV, O’Connor-McCourt M, Pellegrini L, Pelletier J, Shore GC (2011) Mitochondrion-dependent N-terminal processing of outer membrane Mcl-1 protein removes an essential Mule/Lasu1 protein-binding site. J Biol Chem 286:25098–25107PubMedCrossRefGoogle Scholar
  28. 28.
    Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci V, Chiaretti S, Foa R, Schliwka J, Fuchs U, Novosel A, Muller RU, Schermer B, Bissels U, Inman J, Phan Q, Chien M, Weir DB, Choksi R, De Vita G, Frezzetti D, Trompeter HI, Hornung V, Teng G, Hartmann G, Palkovits M, Di Lauro R, Wernet P, Macino G, Rogler CE, Nagle JW, Ju J, Papavasiliou FN, Benzing T, Lichter P, Tam W, Brownstein MJ, Bosio A, Borkhardt A, Russo JJ, Sander C, Zavolan M, Tuschl T (2007) A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129:1401–1414PubMedCrossRefGoogle Scholar
  29. 29.
    Allagnat F, Cunha D, Moore F, Vanderwinden JM, Eizirik DL, Cardozo AK (2011) Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to beta-cell apoptosis. Cell Death Differ 18:328–337PubMedCrossRefGoogle Scholar
  30. 30.
    Chetoui N, Sylla K, Gagnon-Houde JV, Alcaide-Loridan C, Charron D, Al-Daccak R, Aoudjit F (2008) Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis. Mol Cancer Res 6:42–52PubMedCrossRefGoogle Scholar
  31. 31.
    Chan G, Nogalski MT, Bentz GL, Smith MS, Parmater A, Yurochko AD (2010) PI3K-dependent upregulation of Mcl-1 by human cytomegalovirus is mediated by epidermal growth factor receptor and inhibits apoptosis in short-lived monocytes. J Immunol 184:3213–3222PubMedCrossRefGoogle Scholar
  32. 32.
    Morel C, Carlson SM, White FM, Davis RJ (2009) Mcl-1 integrates the opposing actions of signaling pathways that mediate survival and apoptosis. Mol Cell Biol 29:3845–3852PubMedCrossRefGoogle Scholar
  33. 33.
    Son JK, Varadarajan S, Bratton SB (2010) TRAIL-activated stress kinases suppress apoptosis through transcriptional upregulation of MCL-1. Cell Death Differ 17:1288–1301PubMedCrossRefGoogle Scholar
  34. 34.
    Clohessy JG, Zhuang J, de Boer J, Gil-Gomez G, Brady HJ (2006) Mcl-1 interacts with truncated Bid and inhibits its induction of cytochrome c release and its role in receptor-mediated apoptosis. J Biol Chem 281:5750–5759PubMedCrossRefGoogle Scholar
  35. 35.
    Adams JM, Cory S (2007) The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 26:1324–1337PubMedCrossRefGoogle Scholar
  36. 36.
    Thomas LW, Lam C, Edwards SW (2010) Mcl-1; the molecular regulation of protein function. FEBS Lett 584:2981–2989PubMedCrossRefGoogle Scholar
  37. 37.
    Chen ZX, Pervaiz S (2007) Bcl-2 induces pro-oxidant state by engaging mitochondrial respiration in tumor cells. Cell Death Differ 14:1617–1627PubMedCrossRefGoogle Scholar
  38. 38.
    Ahmad KA, Iskandar KB, Hirpara JL, Clement MV, Pervaiz S (2004) Hydrogen peroxide-mediated cytosolic acidification is a signal for mitochondrial translocation of Bax during drug-induced apoptosis of tumor cells. Cancer Res 64:7867–7878PubMedCrossRefGoogle Scholar
  39. 39.
    Low IC, Chen ZX, Pervaiz S (2010) Bcl-2 modulates resveratrol-induced ROS production by regulating mitochondrial respiration in tumor cells. Antioxid Redox Signal 13:807–819PubMedCrossRefGoogle Scholar
  40. 40.
    Howard AN, Bridges KA, Meyn RE, Chandra J (2009) ABT-737, a BH3 mimetic, induces glutathione depletion and oxidative stress. Cancer Chemother Pharmacol 65:41–54PubMedCrossRefGoogle Scholar
  41. 41.
    Pei XY, Dai Y, Grant S (2004) The small-molecule Bcl-2 inhibitor HA14-1 interacts synergistically with flavopiridol to induce mitochondrial injury and apoptosis in human myeloma cells through a free radical-dependent and Jun NH2-terminal kinase-dependent mechanism. Mol Cancer Ther 3:1513–1524PubMedGoogle Scholar
  42. 42.
    Verhaegen M, Bauer JA, Martin de la Vega C, Wang G, Wolter KG, Brenner JC, Nikolovska-Coleska Z, Bengtson A, Nair R, Elder JT, Van Brocklin M, Carey TE, Bradford CR, Wang S, Soengas MS (2006) A novel BH3 mimetic reveals a mitogen-activated protein kinase-dependent mechanism of melanoma cell death controlled by p53 and reactive oxygen species. Cancer Res 66:11348–11359PubMedCrossRefGoogle Scholar
  43. 43.
    Isomoto H, Kobayashi S, Werneburg NW, Bronk SF, Guicciardi ME, Frank DA, Gores GJ (2005) Interleukin 6 upregulates myeloid cell leukemia-1 expression through a STAT3 pathway in cholangiocarcinoma cells. Hepatology 42:1329–1338PubMedCrossRefGoogle Scholar
  44. 44.
    Jourdan M, De Vos J, Mechti N, Klein B (2000) Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-alpha and insulin-like growth factor 1. Cell Death Differ 7:1244–1252PubMedCrossRefGoogle Scholar
  45. 45.
    Wang JM, Lai MZ, Yang-Yen HF (2003) Interleukin-3 stimulation of mcl-1 gene transcription involves activation of the PU.1 transcription factor through a p38 mitogen-activated protein kinase-dependent pathway. Mol Cell Biol 23:1896–1909PubMedCrossRefGoogle Scholar
  46. 46.
    Zhong Q, Gao W, Du F, Wang X (2005) Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis. Cell 121:1085–1095PubMedCrossRefGoogle Scholar
  47. 47.
    Ding Q, He X, Hsu JM, Xia W, Chen CT, Li LY, Lee DF, Liu JC, Zhong Q, Wang X, Hung MC (2007) Degradation of Mcl-1 by beta-TrCP mediates glycogen synthase kinase 3-induced tumor suppression and chemosensitization. Mol Cell Biol 27:4006–4017PubMedCrossRefGoogle Scholar
  48. 48.
    Inuzuka H, Shaik S, Onoyama I, Gao D, Tseng A, Maser RS, Zhai B, Wan L, Gutierrez A, Lau AW, Xiao Y, Christie AL, Aster J, Settleman J, Gygi SP, Kung AL, Look T, Nakayama KI, DePinho RA, Wei W (2011) SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction. Nature 471:104–109PubMedCrossRefGoogle Scholar
  49. 49.
    Wertz IE, Kusam S, Lam C, Okamoto T, Sandoval W, Anderson DJ, Helgason E, Ernst JA, Eby M, Liu J, Belmont LD, Kaminker JS, O’Rourke KM, Pujara K, Kohli PB, Johnson AR, Chiu ML, Lill JR, Jackson PK, Fairbrother WJ, Seshagiri S, Ludlam MJ, Leong KG, Dueber EC, Maecker H, Huang DC, Dixit VM (2011) Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7. Nature 471:110–114PubMedCrossRefGoogle Scholar
  50. 50.
    Su H, Yang JR, Xu T, Huang J, Xu L, Yuan Y, Zhuang SM (2009) MicroRNA-101, down-regulated in hepatocellular carcinoma, promotes apoptosis and suppresses tumorigenicity. Cancer Res 69:1135–1142PubMedCrossRefGoogle Scholar
  51. 51.
    Uhlmann S, Mannsperger H, Zhang JD, Horvat EA, Schmidt C, Kublbeck M, Henjes F, Ward A, Tschulena U, Zweig K, Korf U, Wiemann S, Sahin O (2012) Global microRNA level regulation of EGFR-driven cell-cycle protein network in breast cancer. Mol Syst Biol 8:570PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Jeong-Eun Kwon
    • 1
  • Bu-Yeon Kim
    • 1
  • Seo-Young Kwak
    • 1
  • In-Hwa Bae
    • 1
  • Young-Hoon Han
    • 1
  1. 1.Division of Radiation Cancer ResearchKorea Institute of Radiological and Medical SciencesSeoulSouth Korea

Personalised recommendations