Journal of Endocrinological Investigation

, Volume 42, Issue 11, pp 1307–1317 | Cite as

Expression of miR-217 and HIF-1α/VEGF pathway in patients with diabetic foot ulcer and its effect on angiogenesis of diabetic foot ulcer rats

  • C.-J. LinEmail author
  • Y.-M. Lan
  • M.-Q. Ou
  • L.-Q. Ji
  • S.-D. Lin
Original Article



To investigate the expression of miR-217 and HIF-1α/VEGF pathway in patients with diabetic foot ulcer (DFU) and its effect on angiogenesis in DFU rats.


The serum levels of miR-217, HIF-1α and VEGF were detected in DFU and simple diabetes mellitus (DM) patients, and healthy controls. DFU rat models were established and treated with miR-217 inhibitors and/or HIF-1α siRNA. The ulcer healing of DFU rats was observed. Besides, ELISA method was performed to detect the serum level of HIF-1α, VEGF and inflammatory factors, immunohistochemical (IHC) method to test the micro-vessel density (MVD), as well as qRT-PCR and Western blot to determine expressions of miR-217, HIF-1α, VEGF, VEGFR2, eNOS, MMP-2, and MMP-9 in tissues.


The serum levels of miR-217 were up-regulated while HIF-1α and VEGF were down-regulated in DFU patients and rats when compared with DM and healthy controls (all P < 0.05). Dual-luciferase reporter gene assay confirmed that HIF- was the direct target gene of miR-217. DFU rats treated with miR-217 inhibitors had decreased foot ulcer area and accelerated ulcer healing, with significantly reduced inflammatory factors (IL-1β, TNF-α and IL-6), as well as elevated HIF-1α and VEGF (all P < 0.05); meanwhile, they remarkably increased the MVD in foot dorsum wound tissues and the protein expressions of HIF-1α, VEGF, VEGFR2, eNOS, MMP-2, and MMP-9 (all P < 0.05).


Inhibiting miR-217 could up-regulate HIF-1α/VEGF pathway to promote angiogenesis and ameliorate inflammation of DFU rats, thereby effectively advancing the healing of ulcerated area.


miR-217 HIF-1α VEGF Diabetic foot Angiogenesis 


Compliance with ethical standards

Conflict of interest


Ethical approval

The clinical research in this study was approved by the Ethics Committee of The First Affiliated Hospital of Shantou University Medical College and conformed to the guidelines of Helsinki Declaration.

Informed consent

All samples were collected after obtaining the informed consent form signed by all the subjects.


  1. 1.
    Ahmad J, Zubair M, Malik A, Siddiqui MA, Wangnoo SK (2012) Cathepsin-D, adiponectin, TNF-alpha, IL-6 and hsCRP plasma levels in subjects with diabetic foot and possible correlation with clinical variables: a multicentric study. Foot (Edinb) 22:194–199Google Scholar
  2. 2.
    Aida Y, Maeno M, Suzuki N, Shiratsuchi H, Motohashi M, Matsumura H (2005) The effect of IL-1beta on the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human chondrocytes. Life Sci 77:3210–3221PubMedGoogle Scholar
  3. 3.
    Aumiller WD, Dollahite HA (2015) Pathogenesis and management of diabetic foot ulcers. JAAPA 28:28–34PubMedGoogle Scholar
  4. 4.
    Baptista RB, Souza-Castro N, Almeida-Val VM (2016) Acute hypoxia up-regulates HIF-1alpha and VEGF mRNA levels in Amazon hypoxia-tolerant Oscar (Astronotus ocellatus). Fish Physiol Biochem 42:1307–1318PubMedGoogle Scholar
  5. 5.
    Berchner-Pfannschmidt U, Tug S, Kirsch M, Fandrey J (2010) Oxygen-sensing under the influence of nitric oxide. Cell Signal 22:349–356PubMedGoogle Scholar
  6. 6.
    Botusan IR, Sunkari VG, Savu O, Catrina AI, Grunler J, Lindberg S, Pereira T, Yla-Herttuala S, Poellinger L, Brismar K et al (2008) Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice. Proc Natl Acad Sci USA 105:19426–19431PubMedGoogle Scholar
  7. 7.
    Brem H, Sheehan P, Boulton AJ (2004) Protocol for treatment of diabetic foot ulcers. Am J Surg 187:1S–10SPubMedGoogle Scholar
  8. 8.
    Chen Z, Fu S, Wu Z, Chen J, Huang Y, Wang Y, Fu M (2018) Relationship between plasma angiogenic growth factors and diabetic foot ulcers. Clin Chim Acta 482:95–100PubMedGoogle Scholar
  9. 9.
    de Yebenes VG, Bartolome-Izquierdo N, Nogales-Cadenas R, Perez-Duran P, Mur SM, Martinez N, Di Lisio L, Robbiani DF, Pascual-Montano A, Canamero M et al (2014) miR-217 is an oncogene that enhances the germinal center reaction. Blood 124:229–239PubMedGoogle Scholar
  10. 10.
    Frank RN (2004) Diabetic retinopathy. N Engl J Med 350:48–58PubMedGoogle Scholar
  11. 11.
    Galasso G, Schiekofer S, Sato K, Shibata R, Handy DE, Ouchi N, Leopold JA, Loscalzo J, Walsh K (2006) Impaired angiogenesis in glutathione peroxidase-1-deficient mice is associated with endothelial progenitor cell dysfunction. Circ Res 98:254–261PubMedGoogle Scholar
  12. 12.
    Gao W, Ferguson G, Connell P, Walshe T, Murphy R, Birney YA, O’Brien C, Cahill PA (2007) High glucose concentrations alter hypoxia-induced control of vascular smooth muscle cell growth via a HIF-1alpha-dependent pathway. J Mol Cell Cardiol 42:609–619PubMedGoogle Scholar
  13. 13.
    Gibbons GW (2003) Lower extremity bypass in patients with diabetic foot ulcers. Surg Clin North Am 83:659–669PubMedGoogle Scholar
  14. 14.
    Glynn JJ, Hinds MT (2014) Endothelial outgrowth cells: function and performance in vascular grafts. Tissue Eng Part B Rev 20:294–303PubMedGoogle Scholar
  15. 15.
    Han ZG, Yu TT, Shan L (2012) Expression of erythropoietin and erythropoietin receptor in non-small cell lung cancer and its correlation with microvessel density. Zhonghua Zhong Liu Za Zhi 34:605–608PubMedGoogle Scholar
  16. 16.
    He B, Xiao J, Ren AJ, Zhang YF, Zhang H, Chen M, Xie B, Gao XG, Wang YW (2011) Role of miR-1 and miR-133a in myocardial ischemic postconditioning. J Biomed Sci 18:22PubMedPubMedCentralGoogle Scholar
  17. 17.
    Health USNIo (1985) Laboratory animal welfare: public health service policy on humane care and use of laboratory animals by awardee institutions; notice. Fed Regist 50:19584–19585Google Scholar
  18. 18.
    Hong Q, Li O, Zheng W, Xiao WZ, Zhang L, Wu D, Cai GY, He JC, Chen XM (2017) LncRNA HOTAIR regulates HIF-1alpha/AXL signaling through inhibition of miR-217 in renal cell carcinoma. Cell Death Dis 8:e2772PubMedPubMedCentralGoogle Scholar
  19. 19.
    Jeon O, Song SJ, Bhang SH, Choi CY, Kim MJ, Kim BS (2007) Additive effect of endothelial progenitor cell mobilization and bone marrow mononuclear cell transplantation on angiogenesis in mouse ischemic limbs. J Biomed Sci 14:323–330PubMedGoogle Scholar
  20. 20.
    Keating AM, Jacobs DS (2011) Anti-VEGF treatment of corneal neovascularization. Ocul Surf 9:227–237PubMedGoogle Scholar
  21. 21.
    Kondo T, Ishida Y (2010) Molecular pathology of wound healing. Forensic Sci Int 203:93–98PubMedGoogle Scholar
  22. 22.
    Kuhlencordt PJ, Rosel E, Gerszten RE, Morales-Ruiz M, Dombkowski D, Atkinson WJ, Han F, Preffer F, Rosenzweig A, Sessa WC et al (2004) Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells. Am J Physiol Cell Physiol 286:C1195–C1202PubMedGoogle Scholar
  23. 23.
    Kuwabara M (2016) Hyperuricemia, cardiovascular disease, and hypertension. Pulse (Basel) 3:242–252Google Scholar
  24. 24.
    Ladeia AM, Sampaio RR, Hita MC, Adan LF (2014) Prognostic value of endothelial dysfunction in type 1 diabetes mellitus. World J Diabetes 5:601–605PubMedPubMedCentralGoogle Scholar
  25. 25.
    Lee JH, Gao Z, Ye J (2013) Regulation of 11beta-HSD1 expression during adipose tissue expansion by hypoxia through different activities of NF-kappaB and HIF-1alpha. Am J Physiol Endocrinol Metab 304:E1035–E1041PubMedPubMedCentralGoogle Scholar
  26. 26.
    Lee YC, Hung MH, Liu LY, Chang KT, Chou TY, Wang YC, Wu YC, Lai CL, Tsai CC, Su KC et al (2011) The roles of transforming growth factor-beta(1) and vascular endothelial growth factor in the tracheal granulation formation. Pulm Pharmacol Ther 24:23–31PubMedGoogle Scholar
  27. 27.
    Li N, Luo HC, Yang C, Deng JJ, Ren M, Xie XY, Lin DZ, Yan L, Zhang LM (2014) Cationic star-shaped polymer as an siRNA carrier for reducing MMP-9 expression in skin fibroblast cells and promoting wound healing in diabetic rats. Int J Nanomed 9:3377–3387Google Scholar
  28. 28.
    Li XQ, Chen FS, Tan WF, Fang B, Zhang ZL, Ma H (2017) Elevated microRNA-129-5p level ameliorates neuroinflammation and blood-spinal cord barrier damage after ischemia-reperfusion by inhibiting HMGB1 and the TLR3-cytokine pathway. J Neuroinflammation 14:205PubMedPubMedCentralGoogle Scholar
  29. 29.
    Losi P, Briganti E, Errico C, Lisella A, Sanguinetti E, Chiellini F, Soldani G (2013) Fibrin-based scaffold incorporating VEGF- and bFGF-loaded nanoparticles stimulates wound healing in diabetic mice. Acta Biomater 9:7814–7821PubMedGoogle Scholar
  30. 30.
    Lv Y, Ge L, Zhao Y (2017) Effect and mechanism of SHED on ulcer wound healing in Sprague-Dawley rat models with diabetic ulcer. Am J Transl Res 9:489–498PubMedPubMedCentralGoogle Scholar
  31. 31.
    Manzke E, Katchburian E, Faria FP, Freymuller E (2005) Structural features of forming and developing blood capillaries of the enamel organ of rat molar tooth germs observed by light and electron microscopy. J Morphol 265:335–342PubMedGoogle Scholar
  32. 32.
    Menghini R, Casagrande V, Cardellini M, Martelli E, Terrinoni A, Amati F, Vasa-Nicotera M, Ippoliti A, Novelli G, Melino G et al (2009) MicroRNA 217 modulates endothelial cell senescence via silent information regulator 1. Circulation 120:1524–1532PubMedPubMedCentralGoogle Scholar
  33. 33.
    Moura LI, Dias AM, Carvalho E, de Sousa HC (2013) Recent advances on the development of wound dressings for diabetic foot ulcer treatment—a review. Acta Biomater 9:7093–7114PubMedGoogle Scholar
  34. 34.
    Pakyari M, Farrokhi A, Maharlooei MK, Ghahary A (2013) Critical role of transforming growth factor beta in different phases of wound healing. Adv Wound Care (New Rochelle) 2:215–224Google Scholar
  35. 35.
    Pichu S, Sathiyamoorthy J, Krishnamoorthy E, Umapathy D, Viswanathan V (2015) Impact of the hypoxia inducible factor-1alpha (HIF-1alpha) pro582ser polymorphism and its gene expression on diabetic foot ulcers. Diabetes Res Clin Pract 109:533–540PubMedGoogle Scholar
  36. 36.
    Rahtu-Korpela L, Karsikas S, Horkko S, Blanco Sequeiros R, Lammentausta E, Makela KA, Herzig KH, Walkinshaw G, Kivirikko KI, Myllyharju J et al (2014) HIF prolyl 4-hydroxylase-2 inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction. Diabetes 63:3324–3333PubMedGoogle Scholar
  37. 37.
    Rohani MG, Parks WC (2015) Matrix remodeling by MMPs during wound repair. Matrix Biol 44–46:113–121PubMedGoogle Scholar
  38. 38.
    Saaristo A, Tammela T, Farkkila A, Karkkainen M, Suominen E, Yla-Herttuala S, Alitalo K (2006) Vascular endothelial growth factor-C accelerates diabetic wound healing. Am J Pathol 169:1080–1087PubMedPubMedCentralGoogle Scholar
  39. 39.
    Shao Y, Lv C, Wu C, Zhou Y, Wang Q (2016) Mir-217 promotes inflammation and fibrosis in high glucose cultured rat glomerular mesangial cells via Sirt1/HIF-1alpha signaling pathway. Diabetes Metab Res Rev 32:534–543PubMedGoogle Scholar
  40. 40.
    Shao Y, Ren H, Lv C, Ma X, Wu C, Wang Q (2017) Changes of serum Mir-217 and the correlation with the severity in type 2 diabetes patients with different stages of diabetic kidney disease. Endocrine 55:130–138PubMedGoogle Scholar
  41. 41.
    Shaw TJ, Martin P (2009) Wound repair at a glance. J Cell Sci 122:3209–3213PubMedPubMedCentralGoogle Scholar
  42. 42.
    Su J, Wang Q, Liu Y, Zhong M (2014) miR-217 inhibits invasion of hepatocellular carcinoma cells through direct suppression of E2F3. Mol Cell Biochem 392:289–296PubMedPubMedCentralGoogle Scholar
  43. 43.
    Sun J, Li ZP, Zhang RQ, Zhang HM (2017) Repression of miR-217 protects against high glucose-induced podocyte injury and insulin resistance by restoring PTEN-mediated autophagy pathway. Biochem Biophys Res Commun 483:318–324PubMedGoogle Scholar
  44. 44.
    Sunkari VG, Lind F, Botusan IR, Kashif A, Liu ZJ, Yla-Herttuala S, Brismar K, Velazquez O, Catrina SB (2015) Hyperbaric oxygen therapy activates hypoxia-inducible factor 1 (HIF-1), which contributes to improved wound healing in diabetic mice. Wound Repair Regen 23:98–103PubMedGoogle Scholar
  45. 45.
    Thangarajah H, Vial IN, Grogan RH, Yao D, Shi Y, Januszyk M, Galiano RD, Chang EI, Galvez MG, Glotzbach JP et al (2010) HIF-1alpha dysfunction in diabetes. Cell Cycle 9:75–79PubMedGoogle Scholar
  46. 46.
    Urra H, Hetz C (2014) A novel ER stress-independent function of the UPR in angiogenesis. Mol Cell 54:542–544PubMedGoogle Scholar
  47. 47.
    Wagner FW Jr (1981) The dysvascular foot: a system for diagnosis and treatment. Foot Ankle 2:64–122PubMedGoogle Scholar
  48. 48.
    World Medical A (2013) World medical association declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA 310:2191–2194Google Scholar
  49. 49.
    Xiao H, Gu Z, Wang G, Zhao T (2013) The possible mechanisms underlying the impairment of HIF-1alpha pathway signaling in hyperglycemia and the beneficial effects of certain therapies. Int J Med Sci 10:1412–1421PubMedPubMedCentralGoogle Scholar
  50. 50.
    Yancopoulos GD, Davis S, Gale NW, Rudge JS, Wiegand SJ, Holash J (2000) Vascular-specific growth factors and blood vessel formation. Nature 407:242–248PubMedGoogle Scholar
  51. 51.
    Yazdanpanah L, Nasiri M, Adarvishi S (2015) Literature review on the management of diabetic foot ulcer. World J Diabetes 6:37–53PubMedPubMedCentralGoogle Scholar
  52. 52.
    Zhang EY, Gao B, Shi HL, Huang LF, Yang L, Wu XJ, Wang ZT (2017) 20(S)-Protopanaxadiol enhances angiogenesis via HIF-1alpha-mediated VEGF secretion by activating p70S6 kinase and benefits wound healing in genetically diabetic mice. Exp Mol Med 49:e387PubMedPubMedCentralGoogle Scholar
  53. 53.
    Zhang S, Liu L, Wang R, Tuo H, Guo Y, Yi L, Wang D, Wang J (2013) MicroRNA-217 promotes angiogenesis of human cytomegalovirus-infected endothelial cells through downregulation of SIRT1 and FOXO3A. PLoS One 8:e83620PubMedPubMedCentralGoogle Scholar
  54. 54.
    Zhao QS, Xia N, Zhao N, Li M, Bi CL, Zhu Q, Qiao GF, Cheng ZF (2013) Localization of human mesenchymal stem cells from umbilical cord blood and their role in repair of diabetic foot ulcers in rats. Int J Biol Sci 10:80–89PubMedPubMedCentralGoogle Scholar

Copyright information

© Italian Society of Endocrinology (SIE) 2019

Authors and Affiliations

  • C.-J. Lin
    • 1
    Email author
  • Y.-M. Lan
    • 1
  • M.-Q. Ou
    • 1
  • L.-Q. Ji
    • 1
  • S.-D. Lin
    • 1
  1. 1.Department of Endocrinology and MetabolismThe First Affiliated Hospital of Shantou University Medical CollegeShantouPeople’s Republic of China

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