MicroRNA-33-3p Regulates Vein Endothelial Cell Apoptosis in Selenium-Deficient Broilers by Targeting E4F1

Figure 2

Different miRNA and mRNA expression profiles and qRT-PCR detection and luciferase activity assay. (a) Relative expression levels of miR-33-3p in tissues of the control group and Low-Se group via qRT-PCR. (b) Configuration of the first generation of VECs without treatment. (c) E4F1, a target of miR-33-3p, was predicted by TargetScan software. (d) Wild-type (WT) and mutant-type (MT) (scrambled oligonucleotide) putative miR-33-3p target sequences of E4F1 3TR. (e) To determine the most appropriate transfection concentration in VECs, we transfected different concentrations of miR-33-3p mimic, miR-33-3p inhibitor, the mimic negative control, and the inhibitor negative control for 24 h. miRNA expression of miR-33-3p was detected via qRT-PCR. The optimum concentrations of miR-33-3p mimic and miR-33-3p inhibitor used in this paper were always 300 nM and 100 nM, respectively. (f) Expression of luciferase with the putative miR-33-3p target sites in WT or MT from E4F1 was detected in a luminometer. (g) To prove that transfection is done, efficiency is showed. (h) E4F1 mRNA expression levels in tissues were measured by qRT-PCR. (i) E4F1 mRNA expression levels in VECs were measured by Western blot and ImageJ. shows significant difference (). The results were expressed as the of triplicate cell cultures.