Figure 5. miR-144 directly and indirectly targets c-fos and COX2, and inhibits the synthesis of PGE2.

(A) Luciferase assays were conducted on HeLa cells cotransfected with either miR-144 or mimics NC and the luciferase:c-fos 3′UTR reporter with the WT or mutant miR-144 binding sites. (B) Luciferase assays were conducted on HeLa cells cotransfected with either miR-144 or mimics NC and the luciferase: COX2 3′UTR reporter with the WT or mutant miR-144 binding sites. The luciferase activity in cells cotransfected with miR-144 relative to luciferase activity in cells transfected with the NC are plotted. The mimics NC did not affect reporter activity. The values are presented as the mean ± SD from three independent experiments, each of which was conducted in triplicate. *Statistically significant (P < 0.05) difference compared with cells cotransfected with NC. The results of the cotransfection experiments indicate that miR-144 directly targets the c-fos 3′UTR and the COX2 3′UTR. The effect of miR-144 on c-fos and COX2 was abolished when the putative miR-144 binding sites were mutated. (C–F) WISH cells were transfected with miR-144 mimics, NC mimics, a miR-144 inhibitor or the NC inhibitor. After 48 h, no significant changes in c-fos mRNA levels were observed (C). miR-144 inhibited COX2 mRNA expression (D), reduced c-fos and COX2 protein levels (E), and decreased PGE2 levels (F). (G) miR-144 indirectly regulates COX2 via c-fos. COX2 protein levels increased in cells transfected with miR-144 and the c-fos target protector. (H) miR-144 partially inhibited COX2 expression. WISH cells were transfected with the c-fos overexpression plasmid and either the COX2 target protector or the COX2 target protector NC. Levels of the COX2 protein and PGE2 increased in cells transfected with the c-fos overexpression plasmid and the COX2 target protector (I). The data are presented as the mean ± SD (*P < 0.05).