Figure 1.
Dose dependent flow cytometric detection of cyclobutane pyrimidine dimer (CPD) production after UVA or UVB irradiation in human primary keratinocytes.
Irradiated cells were fixed immediately after irradiation and then labeled with monoclonal antibodies against CPDs. Cellular CPD immuno-fluorescence (FITC) was measured in arbitrary units [a.u.].
Figure 2.
Comparison of UVA and UVB deregulated miRNAs 6h after irradiation.
(A) miRNAs up-regulated (red) or down-regulated (green) after UVA and UVB are colored. (B) Venn diagram shows the overlap between UVA- and UVB-regulated miRNAs. (C) Summary of miRNA analysis of human primary keratinocytes from two female donors after UVA and UVB irradiation. Deregulated miRNAs display a p-value < 0.05 (one sample t-test).
Table 1.
miRNA expression changes in primary human keratinocytes 6h after UVA irradiation (†also changed after UVB).
Table 2.
miRNA expression changes in primary human keratinocytes 6h after UVB irradiation (†also changed after UVA).
Figure 3.
Single primer RT-PCR validation of UVA- and UVB-regulated miRNAs.
The deregulation of selected UV-responsive miRNAs (identified by TaqMan Low Density Arrays) was done by using miRNA specific primer setups followed by the normalization to RNU44.
Figure 4.
Schematic representation of a combination of the most significant networks after UV irradiation using Ingenuity Pathway Analysis of deregulated miRNAs.
(A) The network “Cancer, Endocrine System Disorders, Gastrointestinal Disease” had a highly significant score of 24 after UVA irradiation. (B) After UVB irradiation the network “Reproductive System Disease, Cancer, Cardiovascular Disease” with a score of 35 is shown. (C) Network analysis of miRNAs similarly changed after UVA and UVB radiation identified “Connective Tissue Disorders, Inflammatory Disease, Inflammatory Response” as most significant network (score 14). A solid line represents a direct interaction and a dashed line indicates an indirect interaction. Colored molecules represent miRNAs found to be deregulated in our study (green: down-regulated; red: up-regulated).
Table 3.
Proposed function of the most evidently changed miRNAs after UVA and UVB irradiation.
Figure 5.
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.