miR-128 Is Implicated in Stress Responses by Targeting MAFG in Skeletal Muscle Cells

Figure 1

Validation of miR-128 targets by luciferase assay in HEK293 cells. (a) HEK293 cells were transiently transfected with the luciferase construct bearing wild-type (wt) or inverted (mut) 3UTR fragment holding putative miR-128 target sites of human Nrf2. The alignment of miR-128 with Nrf2-3′UTR is indicated at the top. Cells were simultaneously transfected with either 100 nM of pre-miR-128 (miR-128) or pre-miR negative control (miR-SCR). The pRLSV40 encoding Renilla luciferase plasmid was used as an internal control. Dual luciferase assays were performed as described in the Materials and Methods. Transfections were performed in triplicate, and the ratio (±SD) Firefly/Renilla luciferase activity from three independent experiments were averaged and expressed as percentage of the respective transfections performed with miR-SCR control. . (b) Luciferase constructs containing wild-type (wt) or mutated (mut) 3′UTR of MAFG with predicted miR-128 site (indicated on the top) were cotransfected with either 100 nM of pre-miR-128 (miR-128) or pre-miR negative control (miR-SCR) in HEK293 cells and luciferase activity quantified as described in (a). . (c) Western blotting analysis of MAFG and BMI-1 levels on total proteins from HEK293 cells transfected for 24 h with either 100 nM of pre-miR-128 (miR-128) or pre-miR negative control (miR-SCR); vinculin was used as a loading control.

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