Overexpression of a miR-7-5p mimic does not decrease the abundance of either the endogenous TfR1 mRNA or a luciferase-TfR1 reporter. (A) Overexpression of a miR-7-5p mimic (gray) attenuates the EGFR mRNA, which is a previously identified miR-7-5p target in SW480 cells. A negative control mimic (yellow) with no known microRNA function is also indicated. Cells were harvested after 5 h treatment with 65 µg/mL FAC, similar to the Miyazawa et al. study. The fold change in EGFR mRNA abundance after treatment with the mimic was calculated relative to the nontransfected (vehicle only) control using an HPRT amplicon as the reference (Table 1). (B) The endogenous TfR1 mRNA is not significantly impacted by the same miR-7-5p treatment that attenuates the EGFR mRNA (ns, P = 0.7) (C) Overexpression of the miR-7-5p mimic failed to decrease the activity of the luciferase-TfR1 reporter, as would have been expected if the proposed site were functional. The luciferase reporter with the extended TfR1 3′-UTR (Fig. 2A) was used for the assay. Mutation of the proposed miR-7-5p site (IRE C mut #3) has no impact on the assay, regardless of whether the miR-7-5p mimic is present. Luciferase was measured after 5 h treatment with 65 µg/mL FAC. The activity of the miR-7-5p and negative control mimics is relative to the corresponding minus mimic (vehicle only) transfection. All error bars represent ±SEM of three biological replicates.
