Fig. 3

FTO regulated the miR-3591-5p maturation in an m⁶A-dependent manner base on miRNA-Seq analysis. A Heatmap showed miRNAs expression profile between the mock group, LPS + mock group and LPS + FTO group by miRNA-seq analysis. B The sequences of pre-miR-3591, miR-3591-5p, and the potential m6A motif (AGACU) splicing site were highlighted with different colors. C Chondrocytes were transfected with shFTO or shNC or mock or FTO, and then dealt with 40 ng/mL LPS for 24 h. RT-qPCR was used to assess the mRNA expression pri-miR-3591, pre-miR-3591, and miR-3591-5p. D Chondrocytes were transfected with shFTO or shNC or mock or FTO, and then, the pri-miR-3591 [m⁶A] levels was evaluated using MeRIP coupled with RT-qPCR analysis. E Relative luciferase activity of the wild-type and mutant-type pri-miR-3591 reporter vectors were assessed in FTO knockdown chondrocytes. F RIP combined with RT-qPCR was applied to analyze the pri-miR-3591 binding to DGCR8 in chondrocytes with or without overexpressing FTO. G Quantification of miR-3591-5p, pre-miR-3591, and pri-miR-3591 in the reaction mixture was detected by RT-qPCR, and the results indicated that mutation of [m.⁶A] pri-miR-3591 abolished pri-miR-3591 processing in the in vitro reaction system. LPS, lipopolysaccharide; FTO, FTO overexpression adenovirus; mock, negative control corresponding to FTO overexpression adenovirus; shFTO, FTO knockdown adenovirus; shNC, negative control corresponding to shFTO adenovirus; n = 3, *p < 0.05, **p < 0.01, ***p < 0.001