Fig. 6: Adora2b is a direct target of miR-27b in NHP podocytes.

a Adora2b was induced upon PAN treatment in primary rat podocytes. Rat podocytes were treated with 10 µg/ml PAN for 3 and 5 days before collected for RT-qPCR. Several predicted miR-27b targets from TargetScan with multiple conserved miRNA targeting sites were selected for the analysis. Error bar represents data from two independent experiments. b Adora2b was specifically expressed in rat glomeruli. Primary tissues of glomeruli and tubule were isolated from two different rats. Total RNAs were extracted and expression of Adora2b was analyzed by RT-qPCR. Data were normalized to GAPDH. Error bar represents data from two technical replicates. c Adora2b was enriched in NHP glomeruli. Expression of Adroa2b was analyzed using primary tissues from NHP. Error bar represents data from two technical replicates. d Adora2b and miR-27b were confirmed to be expressed in same glomeruli of NHP by immunohistochemistry and in situ hybridization. FFPE sections of normal monkey kidney tissues were stained with anti-Adora2B and anti-Nephrin antibodies as well as 40 nM microRNA probe. The arrow indicates the same site of Adorae2b, Nephrin, and miR-27b. Scale bar: 25 µm. e Adora2b expression was strongly induced by PAN treatment in NHP podocytes. NHP podocytes were treated with 10 µg/ml PAN for 3–5 days before collected. Upper: protein expression was analyzed by western blotting. Lower: mRNA expression was analyzed by RT-qPCR. Error bar represents data from two independent experiments. f mRNA expression of Adora2b was suppressed by miR-27b in NHP podocytes. Cells were transfected with 50 nM of siRNAs such as siControl/miR-27b mimic, Inhibitor control/miR-27b inhibitors. Total RNAs were extracted for RT-qPCR analysis at 48 h after transfection. Error bar represents data from three independent experiments. *p < 0.05 by Student's t-test. g Protein expression of Adora2b was suppressed by miR-27b in NHP podocytes. Cells were transfected the same way as in f and protein expression of Adora2b was analyzed 48 h post transfection by western blotting. Tubulin was used as the loading control. h Adora2b is a direct target of miR-27b. Adora2b 3′-UTR was cloned into pmiR-glo luciferase reporter (Promega). Dual luciferase assay was done in 293T cells with both reporter and miRNA transfection. miRNA mimic or inhibitor were transfected at 50 nM for 2 days and luciferase activity was analyzed using Dual-Glo assay system (Promega). Error bar represents data from two independent experiments with eight replicates. *p < 0.05 by Student's t-test. i Seed region mutations abolish miR-27b’s suppression on Adora2b. Three point-mutations were introduced into the seed region of each miR-27b target site. These luciferase reporters were then transfected into 293T cells and analyzed in the same way as in g. Error bar represents data from two independent experiments with eight replicates