Repression of let-7 by transforming growth factor-β1-induced Lin28b up-regulates collagen expression in glomerular mesangial cells under diabetic conditions

Abstract

Background: Diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is characterized pathologically by glomerular basement-membrane thickening, mesangial expansion and cellular hypertrophy, and accumulation of extracellular matrix (ECM) proteins such as collagen and fibronectin. Transforming growth factor-β1 (TGF-β1) plays an important role in ECM accumulation in DN. However, to date, the molecular mechanism of TGF-β1 induced ECM accumulation is not fully elucidated. Evidences show that miRNAs play mediatory roles in the progression of DN. Recently, the miRNA let-7 was suggested to be involved as a negative regulator of profibrotic processes in several disease states. In this study, I aimed to investigate whether let-7 directly targets ECM genes to regulate glomerular mesangial fibrosis, and also whether the up-regulation of Lin28, the negative regulator of let-7, is involved in the down-regulation of let-7 in response to TGF-β1 in mesangial cells and under diabetic conditions.Methods: In vitro, mouse glomerular mesangial cells (MMCs) serum depleted for 48 hours and then treated with TGF-β1 for the indicated time periods. TGF-β1 treated MMCs were harvested for RNA and protein isolation to investigate the effect of TGF-β1 on let-7 family miRNAs, target genes, and Lin28b. To evaluate the effect of let-7b regulation on target genes, MMCs were transfected with miRNA mimic or hairpin inhibitor oligonucleotides. Transfected cells were harvested at indicated times for RNA and protein isolation. To verify whether col1a2 and col4a1 are direct targets of let-7b, the col1a2 and col4a1 3′UTRs containing the let-7b binding site were cloned downstream of luciferase in a reporter vector. Luciferase activity was measured in MMCs co-transfected with 3’UTR reporter vectors and let-7b mimic or hairpin inhibitor oligonucleotides. In order to evaluate the role of the potential Smad-binding element (SBE) in TGF-β1 induced Lin28b up-regulation, the proximal promoter and promoter deletion constructs of the mouse Lin28b gene were cloned into a luciferase reporter. Chromatin immunoprecipitation assays were performed to examine whether TGF-β1 activated Smad2/3 binds to the SBE in the proximal promoter with TGF-β1 treatment.In vivo, C57BL/6 mice were injected with 50 mg/kg of streptozotocin intraperitoneally on 5 consecutive days. Control mice were injected with diluent. Each group was comprised of eight mice. All mice were sacrificed at 6 weeks post induction of diabetes. Ten to twelve weeks old type 2 diabetic db/db mice and genetic control non-diabetic db/+ mice were also sacrificed and glomeruli were sieved from renal cortical tissue for RNA extraction.Results: miRNA let-7 family members (let-7b/c/d/g/i) were down-regulated in TGF-β1-treated MMCs along with up-regulation of col1a2 and col4a1. Ectopic expression of let-7b in TGF-β1-treated MMCs attenuated the col1a2 and col4a1 up-regulation. Conversely, let-7b inhibitors increased col1a2 and col4a1 levels. Co-transfection of MMCs with mouse col1a2 or col4a1 3''-UTR luciferase constructs and let-7b inhibitors increased luciferase activity. However, constructs with let-7 target site mutations were un-responsive to TGF-β1. TGF-β1 induced 3''UTR activity was attenuated by let-7b mimics, suggesting col1a2 and col4a1 are direct targets of let-7b. In addition, Lin28b, a negative regulator of let-7 biogenesis, was up-regulated in TGF-β1-treated MMCs. Luciferase assays showed that the Lin28b promoter containing potential Smad-binding site responded to TGF-β1, which was abolished in constructs without the Smad site. Additionally, chromatin immunoprecipitation assays showed TGF-β1-induced enrichment of Smad2/3 at the Lin28 promoter together suggesting that Lin28b is transcriptionally induced by TGF-β1 through the Smad site. Furthermore, let-7b levels were decreased, while Lin28b, col1a2 and col4a1 levels were increased in glomeruli of type 1 and 2 diabetic mice compared to nondiabetic controls, demonstrating in vivo relevance of this Lin28/let-7/collagen axis.Conclusion: In conclusion, these results characterize Lin28b as a new target of TGF-β1 in the kidney. In addition, they suggest a novel role for the Lin28/let-7 pathway in mediating the effects of TGF-β1 on ECM genes in mesangial cells under diabetic conditions.