Accumulation of mesangial extracellular matrix (ECM) proteins such as collagen type 1-a2 (Col1a2) and collagen type 4-a1 (Col4a1) is a key feature of diabetic nephropathy (DN). Transforming growth factor (TGF)-ß1 plays important roles in ECM accumulation in DN, and evidence shows a mediatory role for microRNAs. In the present study, we found that microRNA let-7 family members (let-7b/c/d/g/i) were downregulated in TGF-ß-treated mouse mesangial cells (MMCs) along with upregulation of Col1a2 and Col4a1. Ectopic expression of let-7b in TGF-ß-treated MMCs attenuated Col1a2 and Col4a1 upregulation. Conversely, let-7b inhibitors increased Col1a2 and Col4a1 levels. Cotransfection of MMCs with mouse Col1a2 or Col4a1 3'-untranslated region luciferase constructs and let-7b inhibitors increased luciferase activity. However, constructs with let-7 target site mutations were unresponsive to TGF-ß. TGF-ß-induced 3'-untranslated region activity was attenuated by let-7b mimics, suggesting that Col1a2 and Col4a1 are direct targets of let-7b. In addition, Lin28b, a negative regulator of let-7 biogenesis, was upregulated in TGF-ß-treated MMCs. Lu-ciferase assays showed that the Lin28b promoter containing the Smad-binding element (SBE) responded to TGF-ß, which was abolished in constructs without SBE. Chromatin immunoprecipitation assays showed TGF-ß-induced enrichment of Smad2/3 at the Lin28b promoter, together suggesting that Lin28b is transcriptionally induced by TGF-ß through SBE. Furthermore, let-7b levels were decreased, whereas Lin28b, Col1a2, and Col4a1 levels were increased, in glom-eruli of diabetic mice compared with nondiabetic control mice, demonstrating the in vivo relevance of this Lin28/let-7/collagen axis. These results identify Lin28 as a new TGF-ß target gene and suggest a novel role for the Lin28/let-7 pathway in controlling TGF-ß-induced collagen accumulation in DN.
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