Figure 4. MicroRNA (miR)-195 and miR-497 directly targets CD274. (A) CD274 was detected in MCF7, MDA-MB-231, SK-BR-3 and MCF10A cells by Western blot. (B) Real-time polymerase chain reaction (RT-PCR) was performed to determine miR-195 and miR-497 expression in MCF7, MDA-MB-231, SK-BR-3 and MCF10A. (C) MDA-MB-231 was transfected with miR-195 or miR-497. miR-195, miR-497 and CD274 messenger RNA (mRNA) expression levels were determined via a quantitative RT-PCR assay. (D) MDA-MB-231 was transfected with miR-195 or miR-497. The expression levels of CD274 were determined by Western blotting. (E) CD274 are potential targets of miR-195 and miR-497. The miR-195 and miR-497 target sites and mutant sites in the 3′ untranslated region (3′UTR)s of CD274 are shown. (F) The luciferase vectors that contain the human wild-type (WT) and mutant (MT) CD274 3′UTR regions were co-transfected into MDA-MB-231 cells with miR-195/miR-497 mimic or negative control (NC) miR mimic. The normalized luciferase/Renilla activities were analyzed in the cells 48 hours after the transfection.

CD274=cluster of differentiation; PD-L1=programmed death-ligand 1. ^*p<0.05; ^†p<0.01.