Fig. 6: MiR-34a-5p affects the fibrosis of MCs through Sirt1/HIF-1α signaling.

a Predicted miR-34a-5p target sequence of the Sirt1 3′-UTR showed the highly conserved binding sites in different species. The red nucleotides are the seed sequences of miR-34a-5p. b Dual-luciferase reporter assay was performed to measure the luciferase activity in HEK 293 cells co-transfected with miR-34a-5p and luciferase reporters containing Sirt1 (Sirt1 wt), mutant transcript (Sirt1 mut), or miR-34a-5p inhibitor (as positive control, PC). These results indicated that there was direct interaction between Sirt1 and miR-34a-5p. Data were presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. c, d The mRNA expression level of Sirt1 (c) and HIF-1α (d) regulated by miR-34a-5p mimics or inhibitor was analyzed by qRT-PCR. e–f Protein expression levels of Sirt1 and HIF-1α regulated by miR-34a-5p inhibitor (e) or miR-34a-5p mimics (f) were measured by western blot. g Protein expression levels of HIF-1α, as well as its downstream proteins expression level regulated by combined effects of siRNA-Sirt1 and miR-34a-5p inhibitor were measured by western blot. These results suggested that miR-34a-5p promoted fibrosis in DN might through directly targeting to Sirt1 and regulating the expression of Sirt1. Data were represented as the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01, and ***p < 0.001, n.s: no statistical significance