Fig. 5

Oxidized miR-146a rs2910164 mismatches with the 3’UTRs of IKBA and activates the NF-κB inflammatory pathway. A MiRNA fractions were purified from monocytes isolated from controls and ACS patients, and 8-OHG levels were determined by Northwestern blotting using an anti-8-OHG antibody; B Analysis of 8-OHG in mir-146(C) and mir-146(G) by HPLC–UV spectroscopy; C Outline of luciferase reporter assay for validating the interaction of oxi-miR-146a(G) with IKBA 3′ UTR. The miRNA response elements (MREs) of oxi-miR-146a(G) in human IKBA 3′ UTR were predicted by the bioinformatics program RNAhybrid. The red color indicates the potential mismatching sites in which 8-OHG matches with A. M1, MRE1; M2, MRE2; D Oxidized miR-146a(G) reduces the luciferase activities of IKBA 3′ UTR. HEK293 cells were transfected with the luciferase constructs of the wild-type IKBA 3′ UTR, along with miR-NC, miR-146a(C), miR-146a(G), or oxi-146a(G). The empty vector pGL3 served as a control. ∗ p < 0.05 versus IKBA 3′UTR. The results are expressed as means ± SEM of at least three independent experiments; E–F THP-1 cells were transfected with miR-146a(C) and miR-146a(G), respectively, and then treated with ox-LDL or its control. IκBα(E) and P65 (F) protein levels were analyzed by immunoblot. Quantitative analysis of IκBα and P65 levels is shown in the lower panels. *p < 0.05 and ** p < 0.01 versus Control; G, H P65 immunostaining of atherosclerotic plaque sections of CC, CG, and GG genotypes. Left, overview of plaque; middle and right, higher magnification