Diabetologia

, Volume 57, Issue 5, pp 1037–1046

miR-195 regulates SIRT1-mediated changes in diabetic retinopathy

Article
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Abstract

Aims/hypothesis

Endothelial cell (EC) damage is a key mechanism causing retinal microvascular injury in diabetes. Several microRNAs (miRNAs) have been found to regulate sirtuin 1 (SIRT1, which is involved in regulation of the cell cycle, survival and metabolism) in various tissues and disease states, but no studies have been conducted on the role of miRNA in regulation of SIRT1 in diabetic retinopathy. Here we investigated the effect of miRNA-195 (miR-195), a SIRT1-targeting miRNA, on the development of diabetes-induced changes in ECs and retina.

Methods

The level of miR-195 was measured in human retinal and dermal microvascular ECs (HRECs, HMECs) following exposure to 25 mmol/l glucose (high glucose, HG) and 5 mmol/l glucose (normal glucose, NG). SIRT1 and fibronectin levels were examined following transfection with miR-195 mimic or antagomir or forced expression of SIRT1. Retinal tissues from diabetic rats were similarly studied following intravitreal injection of an miR-195 antagomir or mimic. In situ hybridisation was used to localise retinal miR-195.

Results

HG caused increased miR-195 levels and decreased SIRT1 expression (compared with NG) in both HRECs and HMECs. Transfection with miR-195 antagomir and forced expression of SIRT1 prevented such changes, whereas transfection with miR-195 mimic produced HG-like effects. A luciferase assay confirmed the binding of miR-195 to the 3′ untranslated region of SIRT1. miR-195 expression was upregulated in retinas of diabetic rats and intravitreal injection of miR-195 antagomir ameliorated levels of SIRT1.

Conclusions/interpretation

These studies identified a novel mechanism whereby miR-195 regulates SIRT1-mediated tissue damage in diabetic retinopathy.

Keywords

Diabetic retinopathy Endothelial cell miR-195 SIRT1 

Abbreviations

EC

Endothelial cell

ECM

Extracellular matrix

FN

Fibronectin

HG

High glucose (25 mmol/l d-glucose)

HEK

Human embryonic kidney

HMEC

Human dermal microvascular EC

HREC

Human retinal microvascular EC

miR

microRNA

miRNA

microRNA

MnSOD

Manganese superoxide dismutase

NG

Normal glucose (5 mmol/l d-glucose)

OSM

Osmotic control (25 mmol/l l-glucose)

siRNA

Small interfering RNA

SIRT1

Sirtuin 1; silent information regulator protein 1

STZ

Streptozotocin

3′UTR

3′ Untranslated region

VEGF

Vascular endothelial growth factor

Supplementary material

125_2014_3197_MOESM1_ESM.pdf (106 kb)
ESM Fig. 1MiR-195 regulates glucose-induced SIRT1 mediated aging changes in HMECs. Human dermal microvascular endothelial cells (HMECs) showed glucose-induced (a) miR-195 upregulation (n = 10) and (b) SIRT1 downregulation. (bd) Transfection of HMECs with miR-195 antagomirs normalized HG induced downregulation of SIRT1 mRNA and enzyme activity; (c) shows efficiency of miR-195 antagomir and mimic transfection. n = 6. (e) Senescence associated SA-βGAL staining of HMECs showed increased positivity with 25 mmol/l glucose (HG) treatment compared to 5 mmol/l glucose (NG) and 25 mmol/l l-glucose (OSM). MiR-195 antagomir transfection successfully prevented such signs of cellular aging. Such changes were not seen in cells transfected with scrambled miRNA. Arrow indicates positive cell (blue). (f) Quantitation of SA-βGAL positivity, n = 10 image/treatment. (g) shows HG induced reduction of cellular MnSOD levels were normalized with miR-195 antagomir transfection, n = 6. [Scramble = scrambled miRNAs, 195 = miR-195 mimics, 195(A) = miR-195 antagomirs, * = significantly different from NG, † = significantly different from HG. MiRNA levels are expressed as a ratio to RNU6B (U6); mRNA levels are expressed as a ratio to 18 s. All data are normalized to 5 mmol/l glucose. MnSOD = manganese superoxide dismutase. Scale bar represent 100 μm for all micrographs. Inset = magnified image showing cytoplasmic SA-βGAL positivity.] (PDF 105 kb)
125_2014_3197_MOESM2_ESM.pdf (181 kb)
ESM Fig. 2Plasmid map showing (a) site of SIRT1-3′UTR (wt/mut) insertion in the vector (pMIR-Report-Luciferase plasmid) in regards to the luciferase assay (PDF 180 kb)
125_2014_3197_MOESM3_ESM.pdf (86 kb)
ESM Fig. 3SIRT1 knockdown in NG increases FN protein levels in HRECs. (a) SIRT1 knockdown efficiency with siRNA in NG shows significant reduction of SIRT1 mRNA levels in the ECs and such reduction caused an increase in (b) FN protein levels. Such increase is absent in scramble siRNA treated cells. (c, d) Shows effect of miR-195 antagomir treatment in HRECs in passage 3 has preventative effect against HG induced accelerated aging changes in these cells, n = 10 image/treatment. (e) Shows MnSOD level in HREC passage 3 following miR-195 antagomir transfection. HG induced reduction of MnSOD level is efficiently increased with such treatment in these ECs. [Scramble = scrambled miRNAs. 195(A) = miR-195 antagomirs, HRECs = human retinal microvascular endothelial cells. *=significantly different from NG, †=significantly different from HG. P3 = passage 3, MnSOD = manganese superoxide dismutase. Data normalized to NG. Scale bar represent 100 μm for all micrographs. Inset = magnified image showing cytoplasmic SA-βGAL positivity. n = 6] (PDF 85 kb)
125_2014_3197_MOESM4_ESM.pdf (61 kb)
ESM Fig. 4SIRT1 forced-expression showed preventative effect against glucose-induced damage in HMECs. Transfection of miR-195 antagomir prevented glucose-induced upregulation of (a) FN mRNA and (b) protein levels in the HMECs. (c) Adenoviral forced-expression of SIRT1 in the ECs increased the enzyme’s activity both in NG (5 mmol/l glucose) and HG (25 mmol/l glucose) confirming transfection efficiency at the functional level. Such increase in activity were absent in null vector transfected ECs. Ad-SIRT1 transfected ECs showed (d) attenuated upregulation of HG induced FN mRNA levels and (e) reduced signs of aging with SA-βGAL stain. Arrow indicates positive cell (blue). (f) Quantitation of SA-βGAL positivity, n = 10 image/treatment. [195(A) = miR-195 antagomirs, * = significantly different from NG, † = significantly different from HG; mRNA levels are expressed as a ratio to 18 s normalized to NG. HMECs = human dermal microvascular endothelial cells. All data normalized to controls. Scale bar represent 100 μm for all micrographs. Inset = magnified image showing cytoplasmic SA-βGAL positivity. n = 6] (PDF 60 kb)
125_2014_3197_MOESM5_ESM.pdf (22 kb)
ESM Table 1(PDF 22 kb)

References

  1. 1.
    Khan ZA, Chakrabarti S (2003) Growth factors in proliferative diabetic retinopathy. Exp Diabesity Res 4:287–301PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Antonetti DA, Klein R, Gardner TW (2012) Diabetic retinopathy. N Engl J Med 366:1227–1239PubMedCrossRefGoogle Scholar
  3. 3.
    Mohamed Q, Gillies MC, Wong TY (2007) Management of diabetic retinopathy: a systematic review. JAMA 298:902–916PubMedCrossRefGoogle Scholar
  4. 4.
    Rother KI (2007) Diabetes treatment—bridging the divide. N Engl J Med 356:1499–1501PubMedCrossRefGoogle Scholar
  5. 5.
    Brownlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820PubMedCrossRefGoogle Scholar
  6. 6.
    Joussen AM, Poulaki V, Le ML et al (2004) A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J 18:1450–1452PubMedGoogle Scholar
  7. 7.
    Chen S, Mukherjee S, Chakraborty C, Chakrabarti S (2003) High glucose-induced endothelin-dependent fibronectin synthesis is mediated via NF-kappa B and AP-1. Am J Physiol Cell Physiol 284:263–272CrossRefGoogle Scholar
  8. 8.
    Chen S, Khan ZA, Cukiernik M, Chakrabarti S (2003) Differential activation of NF-kappa B and AP-1 in increased fibronectin synthesis in target organs of diabetic complications. Am J Physiol Endocrinol Metab 284:1089–1097Google Scholar
  9. 9.
    Kaur H, Chen S, Xin X et al (2006) Diabetes-induced extracellular matrix protein expression is mediated by transcription coactivator p300. Diabetes 55:3104–3111PubMedCrossRefGoogle Scholar
  10. 10.
    Chen S, Feng B, George B et al (2010) Transcriptional coactivator p300 regulates glucose-induced gene expression in endothelial cells. Am J Physiol Endocrinol Metab 298:127–137CrossRefGoogle Scholar
  11. 11.
    Chiu J, Khan ZA, Farhangkhoee H, Chakrabarti S (2009) Curcumin prevents diabetes-associated abnormalities in the kidneys by inhibiting p300 and nuclear factor-kappaB. Nutrition 25:964–972PubMedCrossRefGoogle Scholar
  12. 12.
    Xin X, Khan ZA, Chen S, Chakrabarti S (2004) Extracellular signal-regulated kinase (ERK) in glucose-induced and endothelin-mediated fibronectin synthesis. Lab Investig 84:1451–1459PubMedCrossRefGoogle Scholar
  13. 13.
    Roy S, Cagliero E, Lorenzi M (1996) Fibronectin overexpression in retinal microvessels of patients with diabetes. Invest Ophthalmol Vis Sci 37:258–266PubMedGoogle Scholar
  14. 14.
    Khan ZA, Chan BM, Uniyal S et al (2005) EDB fibronectin and angiogenesis—a novel mechanistic pathway. Angiogenesis 8:183–196PubMedCrossRefGoogle Scholar
  15. 15.
    Kumazaki T, Kobayashi M, Mitsui Y (1993) Enhanced expression of fibronectin during in vivo cellular aging of human vascular endothelial cells and skin fibroblasts. Exp Cell Res 205:396–402PubMedCrossRefGoogle Scholar
  16. 16.
    Gama MA, Gasperi RD, Rocher AB et al (2010) Age-related vascular pathology in transgenic mice expressing presenilin 1-associated familial Alzheimer’s disease mutations. Am J Pathol 176:353–368CrossRefGoogle Scholar
  17. 17.
    Sataranatarajan K, Feliers D, Mariappan MM et al (2012) Molecular events in matrix protein metabolism in the aging kidney. Aging Cell 11:1065–1073PubMedCrossRefGoogle Scholar
  18. 18.
    Gerrits PO, Weerd H, Want JJ et al (2010) Microvascular changes in estrogen-α sensitive brainstem structures of aging female hamsters. Neurosci Res 67:267–274PubMedCrossRefGoogle Scholar
  19. 19.
    Sangaralingham SJ, Heublein DM, Grande JP et al (2011) Urinary C-type natriuretic peptide excretion: a potential novel biomarker for renal fibrosis during aging. Am J Physiol Ren Physiol 301:943–952CrossRefGoogle Scholar
  20. 20.
    Jaliffa C, Ameqrane I, Dansault A et al (2009) Sirt1 involvement in rd10 mouse retinal degeneration. Invest Ophthalmol Vis Sci 50:3562–3572PubMedCrossRefGoogle Scholar
  21. 21.
    Brooks CL, Gu W (2009) Opinion: how does SIRT1 affect metabolism, senescence and cancer? Nat Rev Cancer 9:123–128PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Rahman S, Islam R (2011) Mammalian Sirt1: insights on its biological functions. Cell Commun Signal 9:11PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Kubota S, Kurihara T, Ebinuma M et al (2012) Resveratrol prevents light-induced retinal degeneration via suppressing activator protein-1 activation. Am J Pathol 177:1725–1731CrossRefGoogle Scholar
  24. 24.
    Peng CH, Cherng JY, Chiou GY et al (2011) Delivery of Oct4 and SirT1 with cationic polyurethanes-short branch PEI to aged retinal pigment epithelium. Biomaterials 32:9077–9088PubMedCrossRefGoogle Scholar
  25. 25.
    Yamakuchi M (2012) MicroRNA regulation of SIRT1. Front Physiol 3:68PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Mortuza R, Chen S, Feng B, Sen S, Chakrabarti S (2013) High glucose induced alteration of SIRTs in endothelial cells causes rapid aging in a p300 and FOXO regulated pathway. PLoS One 8:e54514PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Chuang JC, Jones PA (2007) Epigenetics and microRNAs. Pediatr Res 61:24–29CrossRefGoogle Scholar
  28. 28.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Ørom UA, Lund AH (2010) Experimental identification of microRNA targets. Gene 451:1–5PubMedCrossRefGoogle Scholar
  30. 30.
    Villeneuve LM, Natarajan R (2010) The role of epigenetics in the pathology of diabetic complications. Am J Physiol Ren Physiol 299:14–25CrossRefGoogle Scholar
  31. 31.
    Lorenzen J, Kumarswamy R, Dangwal S, Thum T (2012) MicroRNAs in diabetes and diabetes-associated complications. RNA Biol 9:820–827PubMedCrossRefGoogle Scholar
  32. 32.
    Wu JH, Gao Y, Ren AJ et al (2012) Altered microRNA expression profiles in retinas with diabetic retinopathy. Ophthalmic Res 47:195–201PubMedCrossRefGoogle Scholar
  33. 33.
    Kantharidis P, Wang B, Carew RM, Lan HY (2011) Diabetes complications: the microRNA perspective. Diabetes 60:1832–1837PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Kovacs B, Lumayag S, Cowan C, Xu S (2011) MicroRNAs in early diabetic retinopathy in streptozotocin-induced diabetic rats. Invest Ophthalmol Vis Sci 52:4402–4409PubMedCrossRefGoogle Scholar
  35. 35.
    Natarajan R, Putta S, Kato M (2012) MicroRNAs and diabetic complications. J Cardiovasc Transl Res 5:413–422PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Karolina DS, Tavintharan S, Armugam A et al (2012) Circulating miRNA profiles in patients with metabolic syndrome. J Clin Endocrinol Metab 97:2271–2276CrossRefGoogle Scholar
  37. 37.
    Chen YQ, Wang XX, Yao XM et al (2011) MicroRNA-195 promotes apoptosis in mouse podocytes via enhanced caspase activity driven by BCL2 insufficiency. Am J Nephrol 34:549–559PubMedCrossRefGoogle Scholar
  38. 38.
    Herrera BM, Lockstone HE, Taylor JM et al (2010) Global microRNA expression profiles in insulin target tissues in a spontaneous rat model of type 2 diabetes. Diabetologia 53:1099–1109PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Zhu H, Yang Y, Wang Y et al (2011) MicroRNA-195 promotes palmitate-induced apoptosis in cardiomyocytes by down-regulating Sirt1. Cardiovasc Res 92:75–84PubMedCrossRefGoogle Scholar
  40. 40.
    Bolmeson C, Esguerra JL, Salehi A et al (2011) Differences in islet-enriched miRNAs in healthy and glucose intolerant human subjects. Biochem Biophys Res Commun 404:16–22PubMedCrossRefGoogle Scholar
  41. 41.
    He JF, Luo YM, Wan XH, Jiang D (2011) Biogenesis of MiRNA-195 and its role in biogenesis, the cell cycle, and apoptosis. J Biochem Mol Toxicol 25:404–408PubMedCrossRefGoogle Scholar
  42. 42.
    McArthur K, Feng B, Wu Y, Chen S, Chakrabarti S (2011) MicroRNA-200b regulates vascular endothelial growth factor-mediated alterations in diabetic retinopathy. Diabetes 60:1314–1323PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Feng B, Chen S, McArthur K et al (2011) miR-146a-mediated extracellular matrix protein production in chronic diabetes complications. Diabetes 60:2975–2984PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Wu Y, Feng B, Chen S, Chakrabarti S (2012) ERK5 regulates glucose-induced increased fibronectin production in the endothelial cells and in the retina in diabetes. Invest Ophthalmol Vis Sci 53:8405–8413PubMedCrossRefGoogle Scholar
  45. 45.
    Turchinovich A, Zoidl, Dermietzel R (2010) Non viral siRNA delivery into the mouse retina in vivo. BMC Ophthalmol 10:25PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Wang M, Tan LP, Dijkstra MK et al (2008) miRNA analysis in B cell chronic lymphocytic leukaemia: proliferation centres characterized by low miR-150 and high BIC/miR-155 expression. J Pathol 215:13–20PubMedCrossRefGoogle Scholar
  47. 47.
    Mimura T, Joyce NC (2006) Replication competence and senescence in central and peripheral human corneal endothelium. Invest Ophthalmol Vis Sci 47:1387–1396PubMedCrossRefGoogle Scholar
  48. 48.
    Jan K, Satoru K, Ravi B et al (2007) Specificity, duplex degradation and subcellular localization of antagomirs. Nucleic Acids Res 35:2885–2892CrossRefGoogle Scholar
  49. 49.
    Giacco F, Brownlee M (2010) Oxidative stress and diabetic complications. Circ Res 107:1058–1070PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Wang R, Zhao N, Li S et al (2013) MicroRNA-195 suppresses angiogenesis and metastasis of hepatocellular carcinoma by inhibiting the expression of VEGF, VAV2, and CDC42. Hepatology 58:642–653PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Rokhsana Mortuza
    • 1
  • Biao Feng
    • 1
  • Subrata Chakrabarti
    • 1
  1. 1.Department of Pathology, Schulich School of Medicine and DentistryWestern UniversityLondonCanada

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