Figure 2.

CRY2 Is a Direct Target of miR-7-5p

(A) A diagram showing candidate miRNAs analyzed by bioinformatics. (B) The mRNA levels of CRY2 detected by qRT-PCR in miR-7-5p overexpression cells. (C) Western blot analyses of CRY2 protein expression in miR-7-5p overexpression cells compared with empty vector. (D) The putative binding sites of miR-7-5p to the 3′ UTR of CRY2 predicted by bioinformatic prediction tools, and the mutation site in the 3′ UTR of CRY2 was shown. (E and F) The effect of miR-7-5p on luciferase activity induced by the pGL3-CRY2-wt and pGL3-CRY2-mut reporter plasmids in HEK293T (E) and C3H10 (F) cells determined by luciferase reporter assays. (G) CRY2 expression in cells with miR-7-5p only or miR-7-5p plus CRY2 cDNA tested by western blot. (H) Quantification of OCN in cells transfected with miR-7-5p only or miR-7-5p plus CRY2 cDNA by qRT-PCR. β-actin is a protein-loading control. All assays were repeated at least three times. The data are the mean ± SD (n = 3). n.s., not significant. *p < 0.05, **p < 0.01, ***p < 0.001.