Identification of direct targets of miR-23b using luciferase assays.

(A) Relative luciferase activities after co-transfection of luciferase constructs and control miRNA or pre-miR-23b in HaCaT cells. The firefly luciferase values were normalized for transfection with renilla luciferase activity. Relative luciferase activities represent the ratio between normalized luciferase activities of pre-miR-23b and control miRNA transfected cells. The mean ± s.e.m. of three independent experiments is shown. (B) Complementarity of miR-23b sequence to the RRAS2 gene sequence. Vertical lines indicate identity between miRNA sequence and corresponding gene sequence. (C) Transcriptional change of the putative targets (RRAS2, TGFBR2 and VHL) of miR-23b in human primary keratinocytes after UVA treatment (600 kJ/m², 6h post irradiation) was analyzed via qPCR. Geometric mean of the expression of the house keeping genes: ACTB (beta actin), HPRT1 (hypoxanthine phosphoribosyltransferase 1) and TBP (TATA box binding protein) was used for normalization. Fold-change of the transcription upon UVA was obtained by setting the control as one-fold. Two-fold threshold was applied as criterion of altered transcriptional response. Error bars indicate standard deviations. N ≥ 3.