RNA-sequencing analysis of the miR-140-CG and -C > T homozygous chondrocytes reveals targets of miR-140 miRNA regulation. (A) Cumulative distribution fraction analysis of expression of predicted miR-140 target genes in miR-140-CG and miR-140-C > T compared to control gene sets. Cumulative distributions of predicted miR-140-target genes with conserved 8-mer binding sites for miR-140-5p, -3p.1, and 3p.2 from miR-140-CG and respective WT chondrocytes and miR-140-C > T and respective WT chondrocytes were plotted based on their expression changes. The black lines indicate distribution of entire genes in these comparisons. The miR-140-5p target gene distribution curve is shifted to the left of the black line, indicating that the expression levels of predicted miR-140-5p target are generally suppressed in -CG or -C > T models. Likewise, miR-140-3p target gene distribution is modestly shifted to the right, demonstrating up-regulation of miR-140-3p target genes in mutant chondrocytes. (B) Two Venn diagrams were created to analyze the overlaps among the deregulated genes in miR-140-CG and miR-140-C > T models, the previously reported miR-140-KO model, and predicted miR-140-5p or miR-140-3p (-3p.1 and -3p.2) targets. Genes whose expression is altered by more than 20.5% were counted. Potential miR-140 targets with conserved 8-mer binding sites were predicted by using TargetScan (v. 7.2). (C) Relative expression of miR-140-5p targets, Wnt11 and Klf9, in -CG and -C > T models. Mean ± SEM, n = 6 from three biological replicates, Student's t-test. (D) Reduced expression of Wnt11 protein in miR-140-CG primary rib chondrocytes. Histone 3 (H3) was used as loading control. The relative signal intensity is indicated. (E) Direct gene regulation by miR-140-5p via the miR-140-5p binding site in the Wnt11 3′-UTR. Luciferase reporter assay was performed in HEK-293-T cells by cotransfecting indicated miRNA mimics with a luciferase reporter construct carrying a wild-type (Wnt11-Wt) or mutated (Wnt11-Mut) miR-140-5p binding site. miR-140-5p, miRIDIAN miR-140-5p mimic; miR-NC, miRIDIAN microRNA mimic negative control #1. n = 6, ANOVA.
