Fig. 8: Silencing of the miR-802 target, Fzd5, impairs insulin gene expression and insulin secretion.

a Si-Fzd5 or over-Fzd5 plasmids was transfected into Min6 cells for 48 h. qRT-PCR was conducted to determine the insulin synthesis, insulin secretion was analyzed by GSIS assay (b) and insulin content was evaluated using ELISA in Min6 cells (c). d miR-802-induced decrease in insulin secretion was partly restored by Fzd5 overexpression. e Overexpression and knockdown miR-802 in Min6 cell by lentivirus, granules were measured by electron microscopy. Scale bars: 20 µm. f Phosphorylation level of CamkII was increased when Fzd5 was overexpressed in Min6 cells. g Overexpression and knockdown of miR-802 in Min6 cells for 48 h; flow cytometry was conducted to investigate the intracellular Ca²⁺ concentration, the numbers represent the percentage of FITC⁺ cells. Ca²⁺ concentration were examined by FLIPR induced by 25 mM glucose (h) and 35 mM KCl (i). j–l Western blot was performed to determine the phosphorylation level of CamkII and CREB in the islets of miR-802 KO mice (j, n = 6, 8–10 weeks old), miR-802 KI mice (k, n = 6, 8–10 weeks old), and len-miR-802 and len-shFoxo1 mice (l, n = 5, 17–18 weeks old). qRT-PCR and western blot were performed to evaluate the mRNA and protein levels of Sox6 in the islet of miR-802 KO mice (m, n = 6, 8–10 weeks old) and miR-802 KI mice (n, n = 6, 8–10 weeks old). o ChIP experiment showed that CREB binds to the promoter of Sox6 by qRT-PCR assays. Only binding site R1 showed positive results. p Min6 cells were co-transfected with Sox6 and insulin promoter luciferase reporter plasmid for 24 h. Relative luciferase activity of the firefly reporter was analyzed. All experiments above were performed in triplicates. Each group contained three batches of individual samples, The p-values by two-tailed unpaired Student’s t test (e, m, n), one-way ANOVA (c, g, f, h, i, p) or two-way ANOVA (a, b, d, o) are indicated. Values are represented as mean ± SD. Source data are provided as a Source Data file.