Fig. 6: Effects of miR-221-3p on high glucose (HG)-induced JAK-STAT signaling in HaCaT cells.

a Venn diagram showing the numbers of the significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways from Gene Set Enrichment Analysis (GSEA, absolute value of normalized enrichment score ≥1.2, P < 0.05) based on RNA sequencing results in the indicated three comparisons. b Intersection of enriched KEGG signaling pathways based on three comparisons were indicated. Filled circle size represents the number of enriched genes from each KEGG pathway (larger circles indicate more enriched genes); circle color, P values (colors closer to blue indicate smaller P values). c Results of GSEA KEGG analysis showing enriched ‘JAK-STAT signaling pathway’. Enrichment plots of ‘JAK-STAT signaling pathway’ from three comparisons were depicted. A positive normalized enrichment score (NES) indicates gene set enrichment at the top of the ranked list, and a negative NES indicates gene set enrichment at the bottom of the ranked list. FDR, false discovery rate. Green line is the enrichment profile. Dark red in the heat map indicates higher gene expression, whereas blue indicates lower gene expression. The gray area of the heat map represents the signal-to-noise ratio of each gene. d, e Representative images and summary data showing the protein levels of phosphorylated (p)-STAT3 (Tyr705), p-STAT3 (Ser727), and total-STAT3. In d, HaCaT cells were cultured in normal glucose (NG, −) or HG (+) medium and transfected with miR-221-3p or miRNA mimic negative control (Negative control). In (e), Ctrl represents miRNA mimic negative control transfection; Mimic, miR-221-3p mimic transfection. Data are presented as mean ± SEM (n = 3–4). *P < 0.05, **P < 0.01 compared with NG-Ctrl; ^##P < 0.01, ^###P < 0.001 for HG-Mimic vs. HG-Ctrl. f, g Representative immunofluorescence images and summary data showing the nuclear localization of STAT3 in HaCaT cells cultured in high glucose (HG) and transfected with miRNA mimic negative control (Ctrl) or miR-221-3p mimic (Mimic) and the ratio of the intensity of STAT3 in the nucleus (Nuc; shown as blue fluorescence with 4’,6-diamidino-2-phenylindole) to that in the cytoplasmic (Cyto; green). Scale bars, 20 μm. Vertically stacked strips of bands in a figure are not derived from the same membrane in any case. Data are presented as mean ± SEM, n = 3. Solid circles represent NG-Ctrl group; squares, NG-Mimic group; equilateral triangles, HG-Ctrl group; and inverted triangles, HG-Mimic group. **P < 0.01 for HG-Ctrl vs. HG-Mimic.