Abstract
MicroRNAs play important roles in the pathogenesis of diabetic nephropathy (DN). In this study, we found that high glucose upregulated miR-27a expression in cultured glomerular mesangial cells and in the kidney glomeruli of streptozotocin (STZ)-induced diabetic rats. miR-27a knockdown prevented high glucose-induced mesangial cell proliferation and also blocked the upregulation of extracellular matrix (ECM)-associated profibrotic genes. Reduction of cell proliferation and profibrotic gene expression by a miR-27a inhibitor depended upon the expression of peroxisome proliferator-activated receptor γ (PPARγ). Further studies showed that miR-27a negatively regulated PPARγ expression by binding to the 3′-untranslated region of rat PPARγ. An antisense oligonucleotide specific to miR-27a (antagomir-27a) significantly reduced renal miR-27a expression in STZ-induced diabetic rats and significantly increased PPARγ levels. Antagomir-27a also reduced kidney ECM accumulation and proteinuria in STZ-induced diabetic rats. These findings suggest that specific reduction of renal miR-27a decreases renal fibrosis, which may be explained in part by its regulation of PPARγ, and that targeting miR-27a may represent a novel therapeutic approach for DN.
Highlights
MicroRNAs are endogenously expressed, small noncoding RNAs that negatively regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of target mRNAs4–6. miRNAs play important roles in cell growth, differentiation, proliferation, apoptosis, and cell death, and contribute to the pathogenesis of many human diseases, including cancer, diabetes, and diabetic complications such as diabetic nephropathy (DN) in addition to other potential diseases[7,8,9]
To examine potential miRNAs that regulate PPARγexpression, we utilized computational prediction programs (TargetScan, PicTar, miRanda, and miRGen) to identify potential binding sites for miRNAs in the peroxisome proliferator-activated receptor γ (PPARγ)-3′-UTR. This analysis indicated that miR-27a has a high probability of binding the 3′-UTR of PPARγmRNA and that the putative miR-27a binding sites in the PPARγ-3′-UTR are highly conserved between several mammals, such as humans, mice, rats, chickens, and dogs (Fig. 1A)
MiR-27a expression was significantly increased in the kidney glomeruli of diabetic rats. These findings suggest that increased miR-27a levels may explain previously reported reductions of PPARγexpression[20,29], which may in turn contribute to DN pathology
Summary
Upregulation of miR-27a expression under hyperglycaemic conditions both in vitro and in vivo. Upregulation of miR-27a by miR-27a mimic increased the levels of these profibrotic genes, compared with NG group These data indicate that mimic and inhibitor of miR-27a had the opposite effect on ECM accumulation. To determine the in vivo relevance of miR-27a knockdown on PPARγexpression in the kidneys of STZ-induced rats, we examined the expression of PPARγmRNA and protein levels in the kidney glomeruli of antagomir-27a-treated rats. In agreement with our in vitro results, the TGF-β1 and PAI-1 levels were reduced in kidney glomeruli samples from antagomir-27a-treated diabetic rats (Fig. 5A–D). Immunohistochemistry revealed that collagen IV and fibronectin levels were significantly attenuated in diabetic rats injected with antagomir-27a (Fig. 5E,F) These results demonstrate that the inhibition of endogenous miR-27a by antagomir-27a and subsequent increase in PPARγlevels triggers the downregulation of several genes that play key profibrotic roles in diabetic rats. These data further support the renoprotective effects of miR-27a inhibition in diabetic rats
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