Figure 4

microRNA-31directly binds to GNA13-3-UTR. (A) Basal GNA13 protein is highly expressed in MDA-MB-231 cells relative to MCF-10a cells. Immunoblot analysis of GNA13 and tubulin expression in MCF-10a and MDA-MB-231 cells is shown. Tubulin is used as a loading control (B) Basal GNA13-3′-UTR activity correlates to GNA13 protein expression. Basal GNA13-3′-UTR reporter activity in MCF-10a and MDA-MB-231 cells was determined. All values are reported relative to to miR-Sens-vector reporter activity in MCF-10a cells. (C) Blockade of miR-31 activity using antimiR-31 rescues GNA13-3′-UTR activity in MCF-10a cells. Reporter assays were performed in MCF-10a cells transfected with antimiR-control or antimiR-31. All values are reported as fold change to miR-Sens-vector in antimiR-control treated cells. (D) Enforced expression of miR-31 in MDA-MB-231 cells suppresses the GNA13-3′-UTR activity in MDA-MB-231 cells. Reporter assays were performed in MDA-MB-231 cells transfected with premiR-control or premiR-31. Results are reported relative to the miR-Sens-vector control treated with premiR-control (*, p < 0.05). miR-31-Sensor is used as a positive control in the reporter assays (C) and (D). This sensor carries a single miR-31 binding site as the 3′-UTR of Renilla Luciferase reporter. Any change in miR-31 levels is efficiently reflected as change in reporter activity. See methods for details.