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. Author manuscript; available in PMC: 2019 Aug 25.
Published in final edited form as: Nat Med. 2019 Feb 25;25(4):583–590. doi: 10.1038/s41591-019-0353-2

Figure 3. Loss-of-function and gain-of-function effects of miR-140 G mutation in chondrocyte transcriptome.

Figure 3.

a, Expression levels of miR-140-5p (WT and G mutant) and two species of miR-140-3p (3p.1 and 3p.2) in primary rib chondrocytes. Rpm: reads per million. Results represent mean ± s.e.m. (n = 3 animals). Statistical significance was assessed using one-way ANOVA and post hoc Tukey-Kramer test. P values indicate comparison versus WT.

b, c, Principle component analysis (b) and hierarchical clustering (c) based on 1000 most variable genes in RNA-seq datasets (n = 3 animals for each group).

d, Cumulative distributions of fold changes of mRNAs with conserved 8mer binding sites for miR-140-5p-WT, -5p-G, -3p.1, and -3p.2 in miR-140 mutant and null chondrocytes. P values (versus a control gene set) were calculated by one-sided Kolmogorov–Smirnov (K-S) test for either direction: upregulation (U) or downregulation (D) with Bonferroni correction (Control genes, n = 11472; miR-140-5p-WT, n = 68; miR-140-5p-G, n = 76; miR-140-3p.1, n = 173; miR-140-3p.2, n = 91).

e, f, Target verification of miR-140-5p-G target sites by 3′ UTR reporter assay in the context of minimal target sites (e) and the endogenous 3′ UTR context (f). Results represent mean ± s.e.m. (n = 3 biologically independent samples). Statistical significance was assessed using two-way ANOVA and post hoc Tukey-Kramer test.

g, Heatmap showing expression changes of representative genes with conserved miR-140-5p-G target sites and downregulation in miR-140G/G chondrocytes. Red denotes important regulators of skeletal development.

h-j, Enrichment map analysis on GSEA results (FDR q-value < 0.1) showing affected cellular programs in miR-140G/G or miR-140-null chondrocytes (h), representative GSEA results for hypoxia-induced gene sets (i, DMOG: hypoxia mimic), and expression heatmap for major Hif1 target genes (j). In (h), enrichments in WT vs. miR-140G/G and WT vs. miR-140-null cells are mapped to the node borders and inner node area, respectively. Red and blue represent upregulation and downregulation in WT cells relative to miR-140G/G or miR-140-null cells, respectively. Pathway enrichment analysis and statistical analysis were performed using GSEA.