Figure 1: MiR-204 is downregulated and Sirt1 is upregulated in aortas of GFM.

(a) Volcano plot of differentially expressed microRNAs in aortas of germ-free mice (GFM). Difference in the expression of aortic microRNAs between GFM and pathogen-free mice (PFM) is plotted on the x axis (log₂ scale), and significance is plotted on the y axis (log₁₀ scale). Upregulated or downregulated microRNAs are indicated in red and blue, respectively. n=3 mice for each microRNA. (b) Decreased mature and precursor miR-204 expression in aortas of GFM by qPCR (shown as % of PFM). n=4 for each group, *P<0.05 versus PFM. (c) In silico analysis of mRNAs targeted by microRNAs that were downregulated by nCounter assay in aortas of GFM. Number of downregulated microRNAs targeting a specific mRNA is shown on the y axis and % of total mRNA is shown on the x axis. MicroRNA-SVR score is colour-coded with threshold<−0.50 in grey, <−0.75 in blue and<−0.90 in red. Lower SVR score indicates stronger predicted microRNA–mRNA interaction. Sirt1 is targeted by microRNAs downregulated in GFM aortas, and based on the number of microRNAs targeting a specific gene it was predicted to be in top 0.5% of putatively regulated genes. (d) MiR-204 mimic (miR-204-M) decreases Sirt1 3′-UTR luciferase reporter activity. SC, scrambled control microRNA. *P<0.05 versus SC, n=5 independent experiments. (e) MiR-204-M (20 nM) decreases Sirt1 expression in human umbilical vein endothelial cells (HUVECs) in a time-dependent manner. Representative of three independent experiments is shown. (f,g) Increased Sirt1 (f) and decreased Trpm3 (g) mRNA expression in aortas of GFM (shown as % of PFM). n=4 for each group, *P<0.05 versus PFM. Independent sample t-test was used. Data shown as mean and error bar represents s.e.m.