miR-383 represses Gadd45g expression by directly targeting Gadd45g 3′-UTR.

<p>(A) Schematic representation of miR-383 binding site on the Gadd45g 3′-UTR. Shaded texts indicate the conserved sequences among human, mouse, rat, rhesus monkey and horse. (B) Gadd45g 3′-UTR sequence containing the predicted target sites was inserted into the pMIR reporter vector, immediately downstream the luciferase gene. The mutant reporter construct was generated by introducing four-mismatch mutation. (C) Relative luciferase activities of Gadd45g 3′-UTR reporter or mutated Gadd45g 3′-UTR reporter in MCF-7 cells with or without miR-383 mimic. Firefly luciferase reading was normalized to that of the Renilla luciferase. Values are means ± SD. (D) MCF-7 cells were co-transfected with the Gadd45g 3′-UTR reporter construct, and anti-miR-383 or anti-control, supplemented by pRL vector, and luciferase activities were analyzed after 48 h. Values are means ± SD. (E) The effect of miR-383 mimic or anti-miR-383 on Gad4d45g protein levels. Protein expression of Gadd45g was determined by western blotting in MCF-7 and MDA-MB-231 cells at 48 h after transfection. β-actin was used as a loading control. (F) Relative Gadd45g mRNA expression was measured by qRT-PCR in MCF-7 and MDA-MB-231 cells transfected with miR-383 mimic or control. Levels were normalized to GAPDH expression. Values are means ± SD.