Figure 3.

Validation of few individual miR-221 target genes. (A) Luciferase assays were performed in Hek293, SNU-398, and HepG2 cells transfected with miR-221 or negative control (NC2), together with psiCheck luciferase vectors containing portions of the 3′UTR of the investigated genes placed downstream the renilla luciferase gene. The renilla luciferase activity was normalized on firefly luciferase activity. Compared to NC2 controls, miR-221 induced a significant decrease in luciferase activity of the vectors containing the RB1, APAF1, ANXA1, WEE1, and CTCF 3′UTR. In contrast, no change in luciferase activity could be detected for the FASLG 3′UTR-luciferase vector and the psiCheck control vector. ^**p-value ≤ 0.01; ^***p-value ≤ 0.001. (B) RB1, APAF1, ANXA1, WEE1, and CTCF mRNA expression was measured in SNU-398 and HepG2 cells following transfection with miR-221 or NC2 using Real Time PCR. A significant reduction in mRNA levels was detected for all the investigated genes, with the exception of CTCF mRNA in HepG2 cells. ^*p-value ≤ 0.05; ^**p-value ≤ 0.01; ^***p-value ≤ 0.001. (C) Protein expression levels were assessed by Western blot analysis. miR-221 induced a decrease in RB1, APAF1, ANXA1, and WEE1 proteins in SNU398 cells, while transfection of anti-miR-221 into SNU-398/miR-221 stable clone induced an increase in target protein levels. Numbers indicate the fold change decrease or increase of proteins expression in miR-221 or anti-miR-221 transfected cells vs. the respective controls. Protein expression levels were normalized vs. β-actin expression.