Figure 4
NF-ĸB1 and IKK-α are targets of miR-15b-5p in colon cancer cells. (A) Schematic representation of the miR-15b-5p putative binding sites in the 3-UTR of NF-ĸB1 or IKK-α mRNA and the mutations introduced into the 3-UTR regions. (B) Wild-type (WT) or mutated type (MT) NF-ĸB1 or IKK-α reporter constructs were co-transfected with miR-15b-5p mimics or NC in HEK293T cells. (C) miR-15b-5p mimic or inhibitor was co-transfected with the pMIR-NFĸB1-3′UTR or pMIR-IKKα-3′UTR vector in HEK293T cells. The relative luciferase activity was measured. (D) NF-ĸB1 and IKK-α protein expression after transfection of miR-15b-5p mimic or inhibitor in SW620 and HCT116 cells. Cells were evaluated at 48 h after transfection by western blotting. Semi-quantitative data from densitometric analysis were presented as the relative ratio of each protein to actin. (E) Representative images of the IHC staining of NF-ĸB1 or IKK-α in 12 colorectal cancer patients. (G) Western blot analysis of anti-apoptotic XIAP, Bcl-xl, and Bcl-2 protein levels in SW620 or HCT116 cells at 48 h after transfection with the miR-15b-5p mimic or a negative control. Actin was used as an internal control. (F) RT-qPCR analysis of relative mRNA expression level in cells at 24 h after transfection with the miR-15b-5p mimic or negative control (*p < 0.05, **p < 0.01, ***p < 0.001).
