Skip to main content
. 2016 Feb;22(2):303–315. doi: 10.1261/rna.052571.115

FIGURE 4.

FIGURE 4.

AXL expression is regulated by microRNAs and through different mechanisms. (A) Identification of the microRNA-binding region of AXL 3′ UTR. The cotransfection reporter assay was performed in the CL1-5 cells using constructs of a series of AXL 3′-UTR deletion mutants (0.5 μg), with Renilla luciferase plasmid serving as an internal control for transfection efficiency. Then the luciferase activity was measured 48 h post-transfection. The schematic presentation on the left shows the deleted regions of various constructs in the AXL 3′ UTR. Reporter activity obtained from the pMiR vector (bottom bar) was set to 1.0 and the activity of the other constructs was calculated and plotted. Relative luciferase activity is shown on the right and is presented as fold values relative to the basal activity. (B) Confirmation of target site (seed region) in AXL 3′ UTR. Luciferase reporter vectors pMIR-Report-AXL 3′ UTR (intact) or pMIR-Report-AXL 3′ UTR (seed region deletion mutant) were transfected into CL1-5 cells, and luciferase activity was measured 24 h later. (C) Inhibition of AXL 3′ UTR by miR-34a/b/c. The AXL 3′-UTR luciferase reporter activities of CL-5 cells transfected with miR-34a/b/c mimics or control oligos were determined. Luciferase activity of control oligos (siRNA) was set as 100% for normalization. An asterisk indicates a significant difference between miR-34a and the control. (D) Expression of the AXL mRNA was determined, in triplicate, by real-time RT-PCR. Fold changes between cells transfected with miR-34a/b/c mimics compared with control oligos are shown. (E) Western blot shows the inhibition of AXL protein levels by miRNAs in CL1-5 cells. Actin serves as a loading control. (F) Discrimination of the target site for miR-34a, but not miR-34c, in AXL 3′ UTR. CL1-5 cells were cotransfected with miR-34a or miR-34c mimic, along with pMIR-Report-AXL 3′ UTR (intact) or pMIR-Report-AXL del 3′ UTR (seed region deletion mutant). An asterisk indicates a significant difference between miR-34c and the control. (G) MiR-34c, but not miR-34a, affects AXL promoter activity. CL-5 cells were transfected with miR-34a or miR-34c mimic along with the pGL3-AXL promoter luciferase construct. Luciferase reporter activity was assayed 48 h later. An asterisk indicates a significant difference between miR-34c and the control. All reporter assays were performed in triplicate and the Renilla reporter activity was used to normalize the transfection efficiency. The error bars represent SD (*) P < 0.05.