, Volume 57, Issue 5, pp 1037–1046

miR-195 regulates SIRT1-mediated changes in diabetic retinopathy




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.


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.


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.


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


Diabetic retinopathy Endothelial cell miR-195 SIRT1 



Endothelial cell


Extracellular matrix




High glucose (25 mmol/l d-glucose)


Human embryonic kidney


Human dermal microvascular EC


Human retinal microvascular EC






Manganese superoxide dismutase


Normal glucose (5 mmol/l d-glucose)


Osmotic control (25 mmol/l l-glucose)


Small interfering RNA


Sirtuin 1; silent information regulator protein 1




3′ Untranslated region


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)


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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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