, Volume 19, Issue 6, pp 975–983 | Cite as

miR-92a inhibits vascular smooth muscle cell apoptosis: role of the MKK4–JNK pathway

  • Lan Zhang
  • Mi Zhou
  • Yingjie Wang
  • Weibin Huang
  • Gangjian Qin
  • Neal L. Weintraub
  • Yaoliang TangEmail author
Original Paper


Vascular smooth muscle cell (VSMC) apoptosis plays an important role in vascular remodeling and atherosclerotic plaque instability. Oxidative stress in diseased vessels promotes VSMC apoptosis in part by activating the c-Jun N-terminal kinase (JNK) pathway, which has been identified as a molecular target of miR-92a in macrophages. Here, we examined the expression and biological activity of miR-92a in VSMC. Quiescent VSMC exhibited a low basal expression of miR-92a, which was positively regulated by serum stimulation and negatively regulated by H2O2. Overexpression of miR-92a decreased H2O2-induced VSMC apoptosis as indicated by TUNEL assay and cleaved caspase-3 protein levels. Using 3′UTR-reporter assay, we found that miR-92a overexpression led to suppression of both mitogen-activated protein kinase kinase 4 (MKK4)- and JNK1-dependent luciferase activity. We also found that 10 mer seed match between miRNA:mRNA pair is more efficient than 8 mer seed match for us to identify authentic miRNA target. Protein levels of active phospho-JNK and phospho-c-Jun, downstream targets of the MKK4–JNK1 pathway, were also decreased by overexpressing miR-92a in VSMC under oxidative stress. Consistent with these findings, overexpression of MKK4 reversed the anti-apoptotic effects of miR-92a in oxidatively stressed VSMC. In conclusion, miR-92a overexpression inhibits H2O2-induced VSMC apoptosis by directly targeting the MKK4–JNK1 pathway.


Vascular smooth muscle cells miR-92a JNK Apoptosis Oxidative stress 



Vascular smooth muscle cells


Mitogen-activated protein kinase


Mitogen-activated protein kinase (MAPK) kinase 4


C-Jun N-terminal kinase



This work was supported by the American Heart Association Beginning Grant-in-Aid 0765094Y (to Y.T.); NIH Grant HL086555 (to Y.T.), and NIH Grants HL076684 and HL62984 (to N.L.W.).

Conflict of interest

None to disclose.

Supplementary material

10495_2014_987_MOESM1_ESM.pdf (619 kb)
Supplementary Figure 1 VSMC were treated with 100 µM H2O2 in DMEM with 0%, 5%, 10% or 20% FBS for 24 h. TUNEL staining (red) showing representative images (A) and quantitative data (B); nuclei are stained with DAPI (PDF 619 kb)


  1. 1.
    Clarke M, Bennett M (2006) Defining the role of vascular smooth muscle cell apoptosis in atherosclerosis. Cell Cycle 5:2329–2331PubMedCrossRefGoogle Scholar
  2. 2.
    Larroque-Cardoso P, Swiader A, Ingueneau C, Negre-Salvayre A, Elbaz M, Reyland ME et al (2013) Role of protein kinase C delta in ER stress and apoptosis induced by oxidized LDL in human vascular smooth muscle cells. Cell Death Dis 4:e520PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Lonn ME, Dennis JM, Stocker R (2012) Actions of “antioxidants” in the protection against atherosclerosis. Free Radic Biol Med 53:863–884PubMedCrossRefGoogle Scholar
  4. 4.
    Murakami T, Takagi H, Suzuma K, Suzuma I, Ohashi H, Watanabe D et al (2005) Angiopoietin-1 attenuates H2O2-induced SEK1/JNK phosphorylation through the phosphatidylinositol 3-kinase/Akt pathway in vascular endothelial cells. J Biol Chem 280:31841–31849PubMedCrossRefGoogle Scholar
  5. 5.
    Kaiser RA, Liang Q, Bueno O, Huang Y, Lackey T, Klevitsky R et al (2005) Genetic inhibition or activation of JNK1/2 protects the myocardium from ischemia-reperfusion-induced cell death in vivo. J Biol Chem 280:32602–32608PubMedCrossRefGoogle Scholar
  6. 6.
    Kim SD, Moon CK, Eun SY, Ryu PD, Jo SA (2005) Identification of ASK1, MKK4, JNK, c-Jun, and caspase-3 as a signaling cascade involved in cadmium-induced neuronal cell apoptosis. Biochem Biophys Res Commun 328:326–334PubMedCrossRefGoogle Scholar
  7. 7.
    Tatsuguchi M, Seok HY, Callis TE, Thomson JM, Chen JF, Newman M et al (2007) Expression of microRNAs is dynamically regulated during cardiomyocyte hypertrophy. J Mol Cell Cardiol 42:1137–1141PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Tang Y, Wang Y, Chen L, Pan Y, Weintraub N (2012) Cross talk between the notch signaling and noncoding RNA on the fate of stem cells. Prog Mol Biol Transl Sci 111:175–193PubMedCrossRefGoogle Scholar
  9. 9.
    Chio CC, Lin JW, Cheng HA, Chiu WT, Wang YH, Wang JJ et al (2013) MicroRNA-210 targets antiapoptotic Bcl-2 expression and mediates hypoxia-induced apoptosis of neuroblastoma cells. Arch Toxicol 87:459–468PubMedCrossRefGoogle Scholar
  10. 10.
    Liu L, Chen R, Huang S, Wu Y, Li G, Zhang B et al (2012) miR-153 sensitized the K562 cells to As2O3-induced apoptosis. Med Oncol 29:243–247PubMedCrossRefGoogle Scholar
  11. 11.
    Qian L, Van Laake LW, Huang Y, Liu S, Wendland MF, Srivastava D (2011) miR-24 inhibits apoptosis and represses bim in mouse cardiomyocytes. J Exp Med 208:549–560PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Cheng Y, Zhang C (2010) MicroRNA-21 in cardiovascular disease. J Cardiovasc Transl Res 3:251–255PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Jazbutyte V, Thum T (2010) MicroRNA-21: from cancer to cardiovascular disease. Curr Drug Targets 11:926–935PubMedCrossRefGoogle Scholar
  14. 14.
    Rippe C, Blimline M, Magerko KA, Lawson BR, LaRocca TJ, Donato AJ et al (2012) MicroRNA changes in human arterial endothelial cells with senescence: relation to apoptosis, eNOS and inflammation. Exp Gerontol 47:45–51PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Iaconetti C, Polimeni A, Sorrentino S, Sabatino J, Pironti G, Esposito G et al (2012) Inhibition of miR-92a increases endothelial proliferation and migration in vitro as well as reduces neointimal proliferation in vivo after vascular injury. Basic Res Cardiol 107:296PubMedCrossRefGoogle Scholar
  16. 16.
    Li WG, Miller FJ Jr, Brown MR, Chatterjee P, Aylsworth GR, Shao J et al (2000) Enhanced H(2)O(2)-induced cytotoxicity in “epithelioid” smooth muscle cells: implications for neointimal regression. Arterioscler Thromb Vasc Biol 20:1473–1479PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Yu SM, Tsai SY, Guh JH, Ko FN, Teng CM, Ou JT (1996) Mechanism of catecholamine-induced proliferation of vascular smooth muscle cells. Circulation 94:547–554PubMedCrossRefGoogle Scholar
  18. 18.
    Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ et al (1995) Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267:682–685PubMedCrossRefGoogle Scholar
  19. 19.
    Whitmarsh AJ, Davis RJ (2007) Role of mitogen-activated protein kinase kinase 4 in cancer. Oncogene 26:3172–3184PubMedCrossRefGoogle Scholar
  20. 20.
    Bonauer A, Carmona G, Iwasaki M, Mione M, Koyanagi M, Fischer A et al (2009) MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science 324:1710–1713PubMedCrossRefGoogle Scholar
  21. 21.
    Lai L, Song Y, Liu Y, Chen Q, Han Q, Chen W et al (2013) MicroRNA-92a negatively regulates Toll-like receptor (TLR)-triggered inflammatory response in macrophages by targeting MKK4 kinase. J Biol Chem 288:7956–7967PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Tchivilev I, Madamanchi NR, Vendrov AE, Niu XL, Runge MS (2008) Identification of a protective role for protein phosphatase 1cgamma1 against oxidative stress-induced vascular smooth muscle cell apoptosis. J Biol Chem 283:22193–22205PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Chaudhry MA, Omaruddin RA, Brumbaugh CD, Tariq MA, Pourmand N (2013) Identification of radiation-induced microRNA transcriptome by next-generation massively parallel sequencing. J Radiat Res 54:808–822PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Ohyashiki M, Ohyashiki JH, Hirota A, Kobayashi C, Ohyashiki K (2011) Age-related decrease of miRNA-92a levels in human CD8 + T-cells correlates with a reduction of naive T lymphocytes. Immun Ageing 8:11PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Rauch C, Feifel E, Amann EM, Spotl HP, Schennach H, Pfaller W et al (2011) Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media. Altex 28:305–316PubMedCrossRefGoogle Scholar
  26. 26.
    Thomas M, Lange-Grunweller K, Hartmann D, Golde L, Schlereth J, Streng D et al (2013) Analysis of transcriptional regulation of the human miR-17-92 cluster; evidence for involvement of Pim-1. Int J Mol Sci 14:12273–12296PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Zheng Z-M, Wang X (2011) Regulation of cellular miRNA expression by human papilloma viruses. Biochim Biophys Acta 1809:668–677PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Woods K, Thomson JM, Hammond SM (2007) Direct regulation of an oncogenic micro-RNA cluster by E2F transcription factors. J Biol Chem 282:2130–2134PubMedCrossRefGoogle Scholar
  29. 29.
    Ohyashiki JH, Umezu T, Kobayashi C, Hamamura RS, Tanaka M, Kuroda M et al (2010) Impact on cell to plasma ratio of miR-92a in patients with acute leukemia: in vivo assessment of cell to plasma ratio of miR-92a. BMC Res Notes 3:347PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Dhanasekaran DN, Reddy EP (2008) JNK signaling in apoptosis. Oncogene 27:6245–6251PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Guma M, Firestein GS (2012) c-Jun N-terminal kinase in inflammation and rheumatic diseases. Open Rheumatol J 6:220–231PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Chadee DN, Kyriakis JM (2010) Activation of SAPK/JNKs in vitro. Methods Mol Biol 661:59–73PubMedCrossRefGoogle Scholar
  33. 33.
    Owen GR, Achilonu I, Dirr HW (2013) High yield purification of JNK1beta1 and activation by in vitro reconstitution of the MEKK1– > MKK4– > JNK MAPK phosphorylation cascade. Protein Expr Purif 87:87–99PubMedCrossRefGoogle Scholar
  34. 34.
    Liu W, Zi M, Jin J, Prehar S, Oceandy D, Kimura TE et al (2009) Cardiac-specific deletion of mkk4 reveals its role in pathological hypertrophic remodeling but not in physiological cardiac growth. Circ Res 104:905–914PubMedCrossRefGoogle Scholar
  35. 35.
    Saha MN, Jiang H, Yang Y, Zhu X, Wang X, Schimmer AD et al (2012) Targeting p53 via JNK pathway: a novel role of RITA for apoptotic signaling in multiple myeloma. PLoS ONE 7:e30215PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Jones EV, Dickman MJ, Whitmarsh AJ (2007) Regulation of p73-mediated apoptosis by c-Jun N-terminal kinase. Biochem J 405:617–623PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Nateri AS, Riera-Sans L, Da Costa C, Behrens A (2004) The ubiquitin ligase SCFFbw7 antagonizes apoptotic JNK signaling. Science 303:1374–1378PubMedCrossRefGoogle Scholar
  38. 38.
    Deng Y, Ren X, Yang L, Lin Y, Wu X (2003) A JNK-dependent pathway is required for TNF alpha-induced apoptosis. Cell 115:61–70PubMedCrossRefGoogle Scholar
  39. 39.
    Nijboer CH, van der Kooij MA, van Bel F, Ohl F, Heijnen CJ, Kavelaars A (2010) Inhibition of the JNK/AP-1 pathway reduces neuronal death and improves behavioral outcome after neonatal hypoxic-ischemic brain injury. Brain Behav Immun 24:812–821PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Lan Zhang
    • 1
    • 5
  • Mi Zhou
    • 2
  • Yingjie Wang
    • 3
  • Weibin Huang
    • 1
  • Gangjian Qin
    • 4
  • Neal L. Weintraub
    • 5
  • Yaoliang Tang
    • 5
    Email author
  1. 1.Department of Vascular Surgery, Renji Hospital, School of MedicineShanghai Jiao Tong UniversityShanghaiChina
  2. 2.Department of Cardiac Surgery, Rui Jin Hospital, School of MedicineShanghai Jiao Tong UniversityShanghaiChina
  3. 3.Internal Medicine of Traditional Chinese MedicineShuguang Hospital of Shanghai University of Traditional Chinese MedicineShanghaiChina
  4. 4.Department of Medicine-Cardiology, Feinberg Cardiovascular Research InstituteNorthwestern University Feinberg School of MedicineChicagoUSA
  5. 5.Department of Medicine, Vascular Biology Center, Medical College of GeorgiaGeorgia Regents UniversityAugustaUSA

Personalised recommendations