Figure 4
ID3 is targeted by miR-10a-3p. (A) A scheme of prediction of potential miR-10a-3p targets. HAND2 and ID3 were the two remaining highly suspected targets of miR-10a-3p used in further analysis. (B) Relative RPKM values of the targets and ossification related genes. *P < 0.05, **P < 0.01, n = 3, t-test. (C) Relative Transcripts Per Million (TPM) values of miR-10a-3p and other identified ossification related miRNAs. *P < 0.05, **P < 0.01, n = 3, t-test. (D) Real-time PCR analysis showing the effect of miR-10a-3p on the RNA level of candidate targets. NC represents overexpression of a scramble Agomir served as negative control. *P < 0.05, n = 3, t-test. (E) Dual luciferase reporter assay of wildtype and mutated (-mut) candidate 3′UTR reporters under miR-10a-3p overexpression. The red squares represent predicted miR-10a-3p target sites, while crossed red square represents mutated target sites. Data were normalized to Renilla luciferase activities and compared to the NC group. **P < 0.01, n = 6, t-test. (F) Western blot analysis of candidate gene products in each treatment group (n = 2), uncropped images were shown in the Supplementary Fig. 5. (G) The effect of miR-10a-3p inhibition on the RNA (left panel) and protein level (right panel) of candidate genes. *P < 0.05, n = 3, t-test. The error bars represents ± S.D.
