Figure 4.

Functional validation of the miR-5003-binding site in the ETS1 3′-UTR and the influence of SNP rs4937333. (A) ETS1 gene structure and T/C polymorphism in the ETS1 3′-UTR at the miR-5003-binding site. chr, chromosome. (B) Binding energy diagram demonstrating the energy transformation of the process of miR-5003 binding to the ETS1 3′-UTR. E_i, E_intermediate; E_t, E_target; E_c, E_complex; ΔE_(a) is the energy difference between the transition state and the original target, and the binding energy, ΔE_(b), is the energy difference between the complex and the target. (C) The effect of the SNP rs4937333 on miR-5003-mediated transcriptional repression in 293T cells. Forty-eight hours after transfection with the reporter gene and miR-5003 mimic, cells with the T allele construct had significantly less relative luciferase activity than in cells with the reporter bearing the C allele. (D) The suppressive effect of miR-5003 on ETS1 gene expression was abolished by rs4937333 C in the ETS1 3′-UTR. ETS1-T or ETS1-C constructs were cotransfected into 293T cells with miR-5003 mimic or miR-NC. ETS1 expression in 293T cells was analyzed by Western blotting at 48 h after transfection. ^∗P < 0.05 vs. miR-NC; n = 3.