Molecular Medicine

, Volume 21, Issue 1, pp 847–860 | Cite as

Downregulation of miRNAs during Delayed Wound Healing in Diabetes: Role of Dicer

  • Sushant Bhattacharya
  • Rangoli Aggarwal
  • Vijay Pal Singh
  • Srinivasan Ramachandran
  • Malabika Datta
Research Article


Delayed wound healing is a major complication associated with diabetes and is a result of a complex interplay among diverse deregulated cellular parameters. Although several genes and pathways have been identified to be mediating impaired wound closure, the role of microRNAs (miRNAs) in these events is not very well understood. Here, we identify an altered miRNA signature in the prolonged inflammatory phase in a wound during diabetes, with increased infiltration of inflammatory cells in the basal layer of the epidermis. Nineteen miRNAs were downregulated in diabetic rat wounds (as compared with normal rat wound, d 7 postwounding) together with inhibited levels of the central miRNA biosynthesis enzyme, Dicer, suggesting that in wounds of diabetic rats, the decreased levels of Dicer are presumably responsible for miRNA downregulation. Compared with unwounded skin, Dicer levels were significantly upregulated 12 d postwounding in normal rats, and this result was notably absent in diabetic rats that showed impaired wound closure. In a wound-healing specific quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) array, 10 genes were significantly altered in the diabetic rat wound and included growth factors and collagens. Network analyses demonstrated significant interactions and correlations between the miRNA predicted targets (regulators) and the 10 wound-healing specific genes, suggesting altered miRNAs might fine-tune the levels of these genes that determine wound closure. Dicer inhibition prevented HaCaT cell migration and affected wound closure. Altered levels of Dicer and miRNAs are critical during delayed wound closure and offer promising targets to address the issue of impaired wound healing.



The authors thank the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for financial help (BSC0302). S Bhattacharya received a fellowship from CSIR.


  1. 1.
    Martin P. (1997) Wound healing: aiming for perfect skin regeneration. Science. 276:75–81.CrossRefGoogle Scholar
  2. 2.
    Broughton G 2nd, Janis JE, Attinger CE. (2006) The basic science of wound healing. Plast. Reconstruct. Surg. 117:12S–34S.CrossRefGoogle Scholar
  3. 3.
    Dovi JV, Szpaderska AM, DiPietro LA. (2004) Neutrophil function in the healing wound: adding insult to injury? Thromb. Haemost. 92:275–80.Google Scholar
  4. 4.
    Koh TJ, DiPietro LA. (2011) Inflammation and wound healing: the role of the macrophage. Expert Rev. Mol. Med. 13:e23.CrossRefGoogle Scholar
  5. 5.
    Singer AJ, Clark RA. (1999) Cutaneous wound healing. N. Engl. J. Med. 341:738–46.CrossRefGoogle Scholar
  6. 6.
    Guo S, Dipietro LA. (2010) Factors affecting wound healing. J. Dent. Res. 89:219–29.CrossRefGoogle Scholar
  7. 7.
    Greenhalgh DG. (2003) Wound healing and diabetes mellitus. Clin. Plast. Surg. 30:37–45.CrossRefGoogle Scholar
  8. 8.
    Falanga V. (2005) Wound healing and its impairment in the diabetic foot. Lancet. 366:1736–43.CrossRefGoogle Scholar
  9. 9.
    Maruyama K, et al. (2007) Decreased macrophage number and activation lead to reduced lymphatic vessel formation and contribute to impaired diabetic wound healing. Am. J. Pathol. 170:1178–91.CrossRefGoogle Scholar
  10. 10.
    Galiano RD, et al. (2004) Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. Am. J. Pathol. 164:1935–47.CrossRefGoogle Scholar
  11. 11.
    Lobmann R, et al. (2002) Expression of matrix-metalloproteinases and their inhibitors in the wounds of diabetic and non-diabetic patients. Diabetologia. 45:1011–16.CrossRefGoogle Scholar
  12. 12.
    Fabian MR, Sonenberg N, Filipowicz W. (2010) Regulation of mRNA translation and stability by microRNAs. Ann. Rev. Biochem. 79:351–79.CrossRefGoogle Scholar
  13. 13.
    Guo H, Ingolia NT, Weissman JS, Bartel DP. (2010) Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature. 466:835–40.CrossRefGoogle Scholar
  14. 14.
    Pandey AK, Agarwal P, Kaur K, Datta M. (2009) MicroRNAs in diabetes: tiny players in big disease. Cell. Physiol. Biochem. 23:221–32.CrossRefGoogle Scholar
  15. 15.
    Sayed D, Abdellatif M. (2011) MicroRNAs in development and disease. Physiol. Rev. 91:827–87.CrossRefGoogle Scholar
  16. 16.
    Soifer HS, Rossi JJ, Saetrom P. (2007) MicroRNAs in disease and potential therapeutic applications. Mol. Ther. 15:2070–79.CrossRefGoogle Scholar
  17. 17.
    Funari VA, et al. (2013) Differentially expressed wound healing-related microRNAs in the human diabetic cornea. PLoS One. 8:e84425.CrossRefGoogle Scholar
  18. 18.
    Pastar I, et al. (2011) Micro-RNAs: new regulators of wound healing. Surg. Technol. Int. 21:51–60.PubMedGoogle Scholar
  19. 19.
    Biswas S, et al. (2010) Hypoxia inducible microRNA 210 attenuates keratinocyte proliferation and impairs closure in a murine model of ischemic wounds. Proc. Natl. Acad. Sci. U. S. A. 107:6976–81.CrossRefGoogle Scholar
  20. 20.
    Chan YC, Roy S, Khanna S, Sen CK. (2012) Downregulation of endothelial microRNA-200b supports cutaneous wound angiogenesis by desilencing GATA binding protein 2 and vascular endothelial growth factor receptor 2. Arterioscler. Thromb. Vasc. Biol. 32:1372–82.CrossRefGoogle Scholar
  21. 21.
    Pastar I, et al. (2012) Induction of specific microRNAs inhibits cutaneous wound healing. J. Biol. Chem. 287:29324–35.CrossRefGoogle Scholar
  22. 22.
    Xu J, et al. (2012) The role of microRNA-146a in the pathogenesis of the diabetic wound-healing impairment: correction with mesenchymal stem cell treatment. Diabetes. 61:2906–12.CrossRefGoogle Scholar
  23. 23.
    Madhyastha R, Madhyastha H, Nakajima Y, Omura S, Maruyama M. (2012) MicroRNA signature in diabetic wound healing: promotive role of miR-21 in fibroblast migration. Int. Wound J. 9:355–61.CrossRefGoogle Scholar
  24. 24.
    Sundaram GM, et al. (2013) ‘See-saw’ expression of microRNA-198 and FSTL1 from a single transcript in wound healing. Nature. 495:103–6.CrossRefGoogle Scholar
  25. 25.
    Eming SA, Krieg T, Davidson JM. (2007) Inflammation in wound repair: molecular and cellular mechanisms. J. Invest. Dermatol. 127:514–25.CrossRefGoogle Scholar
  26. 26.
    Menke NB, Ward KR, Witten TM, Bonchev DG, Diegelmann RF. (2007) Impaired wound healing. Clin. Dermatol. 25:19–25.CrossRefGoogle Scholar
  27. 27.
    Pfaffl MW. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res. 29:e45.CrossRefGoogle Scholar
  28. 28.
    Saito R, et al. (2012) A travel guide to Cytoscape plugins. Nat. Methods. 9:1069–76.CrossRefGoogle Scholar
  29. 29.
    Grice EA, et al. (2010) Longitudinal shift in diabetic wound microbiota correlates with prolonged skin defense response. Proc. Natl. Acad. Sci. U. S. A. 107:14799–804.CrossRefGoogle Scholar
  30. 30.
    Galiano RD, et al. (2004) Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells. Am. J. Pathol. 164:1935–47.CrossRefGoogle Scholar
  31. 31.
    Maehr R, et al. (2005) Cathepsin L is essential for onset of autoimmune diabetes in NOD mice. J. Clin. Invest. 115:2934–43.CrossRefGoogle Scholar
  32. 32.
    Lan CE, Wu C, Huang S, Wu I, Chen G. (2013) High-glucose environment enhanced oxidative stress and increased interleukin-8 secretion from keratinocytes. Diabetes. 62:2530–8.CrossRefGoogle Scholar
  33. 33.
    Wang XJ, Han G, Owens P, Siddiqui Y, Li AG. (2006) Role of TGF beta-mediated inflammation in cutaneous wound healing. J. Invest. Dermatol. 11:112–17.CrossRefGoogle Scholar
  34. 34.
    Holmes C, Wrobel JS, Maceachern MP, Boles BR. (2013) Collagen-based wound dressings for the treatment of diabetes-related foot ulcers: a systematic review. Diabetes Metab. Syndr. Obes. 6:17–29.CrossRefGoogle Scholar
  35. 35.
    Ebaid H, Ahmed OM, Mahmoud AM, Ahmed RR. (2013) Limiting prolonged inflammation during proliferation and remodeling phases of wound healing in streptozotocin-induced diabetic rats supplemented with camel undenatured whey protein. BMC Immunol. 14:31.CrossRefGoogle Scholar
  36. 36.
    Bertero T, et al. (2011) miR-483-3p controls proliferation in wounded epithelial cells. FASEB J. 25:3092–105.CrossRefGoogle Scholar
  37. 37.
    Shilo S, Roy S, Khanna S, Sen CK. (2007) MicroRNA in cutaneous wound healing: a new paradigm. DNA Cell Biol. 26:227–37.CrossRefGoogle Scholar
  38. 38.
    Szczepankiewicz A, Lackie PM, Holloway JW. (2013) Altered microRNA expression profile during epithelial wound repair in bronchial epithelial cells. BMC Pulm. Med. 13:63.CrossRefGoogle Scholar
  39. 39.
    Lee Y, et al. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature. 425:415–9.CrossRefGoogle Scholar
  40. 40.
    Andl T, et al. (2006) The miRNA-processing enzyme dicer is essential for the morphogenesis and maintenance of hair follicles. Curr. Biol. 16:1041–9.CrossRefGoogle Scholar
  41. 41.
    Schultz HY, Goldsmith LA. (2007) Looking ahead by looking back. J. Invest. Dermatol. 127:1–2.CrossRefGoogle Scholar
  42. 42.
    Yi R, et al. (2006) Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs. Nat. Genet. 38:356–62.CrossRefGoogle Scholar
  43. 43.
    Banerjee J, Chan YC, Sen CK. (2011) MicroRNAs in skin and wound healing. Physiol. Genomics. 43:543–56.CrossRefGoogle Scholar
  44. 44.
    Ghatak S, et al. (2015) Barrier function of the repaired skin is disrupted following arrest of Dicer in keratinocytes. Mol. Ther. 23:1201–10.CrossRefGoogle Scholar
  45. 45.
    Mori MA, et al. (2012) Role of MicroRNA processing in adipose tissue in stress defense and longevity. Cell Metab. 16:336–47.CrossRefGoogle Scholar
  46. 46.
    Suarez Y, et al. (2008) Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis. Proc. Natl. Acad. Sci. U. S. A. 105:14082–87.CrossRefGoogle Scholar
  47. 47.
    Olczyk P, Mencner L, Komosinska-Vassev K. (2014) The role of the extracellular matrix components in cutaneous wound healing. Biomed. Res. Int. 2014:747584.Google Scholar
  48. 48.
    Cullen B, et al. (2002) The role of oxidised regenerated cellulose/collagen in chronic wound repair and its potential mechanism of action. Int. J. Biochem. Cell Biol. 34:1544–56.CrossRefGoogle Scholar
  49. 49.
    Lee CH, Shah B, Moioli EK, Mao JJ. (2010) CTGF directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model. J. Clin. Invest. 120:3340–49.CrossRefGoogle Scholar
  50. 50.
    Ivkovic S, et al. (2003) Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development. 130:2779–91.CrossRefGoogle Scholar
  51. 51.
    Igarashi A, Okochi H, Bradham DM, Grotendorst GR. (1993) Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol. Biol. Cell. 4:637–45.CrossRefGoogle Scholar
  52. 52.
    Li AG, Wang D, Feng XH, Wang XJ. (2004) Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder. EMBO J. 23:1770–81.CrossRefGoogle Scholar
  53. 53.
    O’Kane S, Ferguson MW. (1997) Transforming growth factor beta s and wound healing. Int. J. Biochem. Cell Biol. 29:63–78.CrossRefGoogle Scholar
  54. 54.
    Badr G, et al. (2012) Treatment of diabetic mice with undernatured whey protein accelerates the wound healing process by enhancing the expression of MIP-1alpha, MIP-2, KC, CX3CL1 and TGF-beta in wounded tissue. BMC Immunol. 13:32.CrossRefGoogle Scholar
  55. 55.
    Liu X, et al. (2001) Conditional epidermal expression of TGFbeta 1 blocks neonatal lethality but causes a reversible hyperplasiaand alopecia. Proc. Natl. Acad. Sci. U. S. A. 98:9139–44.CrossRefGoogle Scholar
  56. 56.
    Liu X, et al. (1997) Transforming growth factor beta-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc. Natl. Acad. Sci. U. S. A. 94:10669–74.CrossRefGoogle Scholar
  57. 57.
    Emmerson E, et al. (2012) Insulin-like growth factor-1 promotes wound healing in estrogendeprived mice: new insights into cutaneous IGF-1R/ERα cross talk. J. Invest. Dermatol. 132:2838–48.CrossRefGoogle Scholar
  58. 58.
    Bos PK, van Osch GJ, Frenz DA, Verwoerd-Verhoef HL. (2001) Growth factor expression in cartilage wound healing: temporal and spatial immunolocalization in a rabbit auricular cartilage wound model. Osteoarthritis Cartilage. 9:382–9.CrossRefGoogle Scholar
  59. 59.
    Gartner MH, Benson JD, Caldwell MD. (1992) Insulin-like growth factors I and II expression in the healing wound. J. Surg. Res. 52:389–94.CrossRefGoogle Scholar
  60. 60.
    Todorovic V, et al. (2008) Insulin-like growth factor-I in wound healing of rat skin. Regul. Pept. 150:7–13.CrossRefGoogle Scholar
  61. 61.
    Toulon A, et al. (2009) A role for human skinresident T cells in wound healing. J. Exp. Med. 206:743–50.CrossRefGoogle Scholar
  62. 62.
    Telasky C, et al. (1998) IFN-alpha2b suppresses the fibrogenic effects of insulin-like growth factor-1 in dermal fibroblasts. J. Interferon Cytokine Res. 18:571–7.CrossRefGoogle Scholar
  63. 63.
    Haase I, Evans R, Pofahl R, Watt FM. (2003) Regulation of keratinocyte shape, migration and wound epithelialization by IGF-1- and EGF-dependent signalling pathways. J. Cell Sci. 116:3227–38.CrossRefGoogle Scholar
  64. 64.
    Blakytny R, Jude EB, Martin Gibson J, Boulton AJ, Ferguson MW. (2000) Lack of insulin-like growth factor 1 (IGF1) in the basal keratinocyte layer of diabetic skin and diabetic foot ulcers. J. Pathol. 190:589–94.CrossRefGoogle Scholar
  65. 65.
    Brown DL, Kane CD, Chernausek SD, Greenhalgh DG. (1997) Differential expression and localization of insulin-like growth factors I and II in cutaneous wounds of diabetic and nondiabetic mice. Am. J. Pathol. 151:715–24.PubMedPubMedCentralGoogle Scholar
  66. 66.
    Bitar MS. (1997) Insulin-like growth factor-1 reverses diabetes-induced wound healing impairment in rats. Horm. Metab. Res. 29:383–6.CrossRefGoogle Scholar
  67. 67.
    Hirsch T, et al. (2008) Insulin-like growth factor-1 gene therapy and cell transplantation in diabetic wounds. J. Gene Med. 10:1247–52.CrossRefGoogle Scholar
  68. 68.
    Grøndahl-Hansen J, Lund LR, Ralfkiaer E, Ottevanger V, Danø K. (1988) Urokinase- and tissue-type plasminogen activators in keratinocytes during wound reepithelialization in vivo. J. Invest. Dermatol. 90:790–5.CrossRefGoogle Scholar
  69. 69.
    Lund LR, et al. (2006) Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice. EMBO J. 25:2686–97.CrossRefGoogle Scholar
  70. 70.
    Rømer J, et al. (1996) Impaired wound healing in mice with a disrupted plasminogen gene. Nat. Med. 2:287–92.CrossRefGoogle Scholar
  71. 71.
    Miragliotta V, Ipiña Z, Lefebvre-Lavoie J, Lussier JG, Theoret CL. (2008). Equine CTNNB1 and PECAM1 nucleotide structure and expression analyses in an experimental model of normal and pathological wound repair. BMC Physiol. 8:1.CrossRefGoogle Scholar
  72. 72.
    Cheon S, et al. (2005) Prolonged beta-catenin stabilization and tcf-dependent transcriptional activation in hyperplastic cutaneous wounds. Lab. Invest. 85:416–25.CrossRefGoogle Scholar
  73. 73.
    Soler C, Grangeasse C, Baggetto LG, Damour O. (1999) Dermal fibroblast proliferation is improved by beta-catenin overexpression and inhibited by E-cadherin expression. FEBS Lett. 442:178–82.CrossRefGoogle Scholar
  74. 74.
    Cheon S, et al. (2002) Beta-catenin stabilization dysregulates mesenchymal cell proliferation, motility, and invasiveness and causes aggressive fibromatosis and hyperplastic cutaneous wounds. Proc. Natl. Acad. Sci. U. S. A. 99:6973–8.CrossRefGoogle Scholar
  75. 75.
    Amini-Nik S, et al. (2014) β-Catenin-regulated myeloid cell adhesion and migration determine wound healing. J. Clin. Invest. 124:2599–610.CrossRefGoogle Scholar
  76. 76.
    Desai LP, Aryal AM, Ceacareanu B, Hassid A, Waters CM. (2004) RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 287:L1134–44.CrossRefGoogle Scholar

Copyright information

© The Author(s) 2015

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (

Authors and Affiliations

  • Sushant Bhattacharya
    • 1
  • Rangoli Aggarwal
    • 1
  • Vijay Pal Singh
    • 1
  • Srinivasan Ramachandran
    • 1
  • Malabika Datta
    • 1
  1. 1.Council of Scientific and Industrial Research (CSIR)Institute of Genomics and Integrative BiologyDelhiIndia

Personalised recommendations