Figure 3
IGF1R, IL-6R and IL-6 are direct targets of let-7c/miR-99a/miR-125b. (a) The positive correlation among miR-99a, let-7c and miR-125b, correlation analysis was determined using the Pearson product–moment correlation. (b) Schematic representation of the interaction of an miRNA with the 3′ UTR of its corresponding target. Each predicted MRE 3′ UTR was inserted into a psiCHECK-2 vector immediately downstream from the Renilla luciferase gene. Luciferase reporter assays analyzing the putative targets of the three miRNAs. HEK-293T cells were co-transfected with psiCHECK-2 plasmids with the 3′ UTR of the target genes or the target genes with mutated miRNA-binding sites (Mut) and miRNA mimics or negative control mimics (NC) using Lipofectamine 2000. Firefly luciferase activity was normalized to Renilla luciferase activity, and the results were expressed relative to the control. (c) QBC939, MZ-cha-1 and SK-cha-1 cells were infected by lentivirus overexpressing miRNA or negative controls, and cells lysates were prepared for western blotting with antibodies against IGF1R, IL-6R or IL-6. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression served as a loading control, and densitometric ratios were recorded. Experiments were performed in triplicate, and all indicated P-values were determined using t-test. *P<0.05; **P<0.01.
