Figure 2. miR-125c directly targets cdc25a which responds to cellular hypoxia.

(A) Schematic illustration of cdc25a 3′UTR fragment harboring a miR-125c binding site (highlighted in italics). The sequence information of the cdc25a 3′UTR in wild type (WT) and its mutant (Mut) with disrupted base pairing are indicated. (B) Dual-luciferase reporter assay for validation of miR-125c binding site on the 3′UTR of cdc25a. HeLa cells were cotransfected with cdc25a 3′UTR dual-luciferase constructs (WT or Mut) plus miR-125c mimics. Renilla luciferase activities were detected and normalized to Firefly luciferase (internal control). Results represent means ± S.E. (n = 3, **P < 0.01). (C) Transfection efficiency of miR-125c mimics and inhibitor in ZF4 cells was detected by qRT-PCR. All data are means ± S.D. (n = 3, **P < 0.01). (D) The cdc25a expression in ZF4 cells transfected with miR-125c mimics or inhibitor was detected by qRT-PCR. 18s rRNA is used as the endogenous control. Values represent means ±S.D. (n = 3, *P < 0.05). (E) Expression analysis of Cdc25a protein by western blot in corresponding ZF4 cell samples. β-actin is used to normalize protein levels. Numbers indicate quantification of the Cdc25a band densities relative to β-actin. (F) Injection efficiency of miR-125c mimics in zebrafish embryos was detected by qRT-PCR. U6-1 is used as the endogenous control. Values represent means ±S.D. (n = 3, **P < 0.01). (G) The regulation of cdc25a expression by miR-125c in zebrafish embryos based on qRT-PCR. 18 s rRNA is used as the endogenous control. Values represent means ±S.D. (n = 3, **P < 0.01). (H) The regulation of Cdc25a protein expression by miR-125c in zebrafish embryos based on western blot analysis. β-actin is used to normalize protein levels. Numbers indicate quantification of the Cdc25a band densities relative to β-actin. (I) Expression of cdc25a in ZF4 cells treated with 100 µM CoCl₂ for 0 h, 8 h and 12 h before being collected. 18 s rRNA is used as the endogenous control. Values represent means ±S.D. (n = 3, **P < 0.01). (J) Expression analysis of Cdc25a protein by western blot in corresponding ZF4 cell samples. β-actin is used to normalize protein levels. Numbers indicate quantification of the Cdc25a band densities relative to β-actin.