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Subrata Chakrabarti, Anu Thomas, Saumik Biswas, Biao Feng, Shali Chen, John Gonder; Novel Long non-coding RNA mediated EndMT pathway in the retina in diabetes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2657.
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We have previously shown that endothelial-mesenchymal transition (EndMT) is mediated by miR-200b in the retina in diabetes. Long non-coding RNAs (lncRNAs) influence various diseases. lncRNA H19 regulates members of miR-200 family in other systems. We examined retinas for H19-mediated regulation of EndMT directly or through miR200b in diabetes.
Retinal endothelial cells were treated with high (25mM,HG) or normal glucose (5mM) for various duration. Transfection with H19 siRNA and H19 overexpressing vector were followed by RNA analyses. We investigated retinal tissues from STZ-induced diabetic mice with or without H19 knockout and human vitreous samples. We also used a pcDNA 3.1(+) harboring a full-length cDNA to overexpress human H19 as well as miR200b mimics and antagomirs.
HG treatment showed reduced endothelial markers; CD-31 and VE-cad and increased mesenchymal markers; FSP1, SM22 and α-SMA confirming EndMT. These changes were prevented by miR-200b mimic transfection. H19 overexpression increased basal and glucose-induced miR-200b downregulation. H19 expression was unaffected by 200b overexpression. To outline a direct relationship, we performed rescue using miR-200b antagomirs and overexpressed H19. Such intervention prevented glucose-induced EndMT. In all instances, HG induced EndMT protein changes were corrected by H19 overexpression. Diabetic wild-type and H19 knockout mice and their controls were monitored for 2 months. Retinal RNA from diabetic animals and human vitreous showed H19 reductions. Retinal EndMT was seen in diabetic animals and in H19KO mice. We investigated TGFβ pathway, key regulator of EndMT. HG caused increased production or phosphorylation of TGFβRII (total), pSmad 2, pSmad 3 and Smad 4 (total), pAKT and pERK1/2 proteins. TGFβRII (total) was significantly reduced following H19 overexpression Basal levels of SMAD proteins (pSmad 2, pSmad 3, Smad 4) and pAKT were increased following H19 overexpression. However, levels of these molecules in the HG appeared to be unaltered following H19 overexpression. Interestingly, pERK1/2 levels were significantly downregulated in samples overexpressing H19 irrespective of glucose levels.
This study showed that glucose-induced H19 downregulation aided EndMT. Such effects are possibly mediated by miR-200b through regulation of Smad-independent MAP-ERK 1/2 pathway of TGF-β signaling..
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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