Fig. 5
DIAPH2 is a direct downstream target of miR-10b in RDEB-cSCC. a Kaplan-Meier curves of the metastatic HNSCC (TCGA RNAseq and clinical data filtered for stage IV, N3). Data show significant (p = 0.024, log-rank test) impact of DIAPH2 expression (75% quantile is considered for stratification between high and mid/low expression) on mean survival time (mst) mst DIAPH2high = 2166 vs. mst DIAPH2low = 806 days. b Overall expression of DIAPH2 in cultured cells was significantly (p < 0.001) reduced in RDEB-cSCC (n = 3) compared to primary keratinocytes. No significant difference was found between HC-KC and RDEB-KC in dosimetric Western blot analysis (data represented by mean ± standard deviation of three independent experiments, unpaired t-test). c Significant (p = 0.045) lower DIAPH2 protein levels appeared in one out of three HC-cSCC cell lines in relation to HC-KC (1090KC, mean ± SEM of three ind. experiments, unpaired t-test). d Immunofluorescence microscopy confirmed reduced DIAPH2 levels in one out or three HC-cSCC and all three RDEB-cSCC cell lines (2 independent repeats). e Western blot analysis shows recurrence of DIAPH2 expression in an RDEB-SCC1MIR10B−/− culture generated by serial dilution (data represented by mean ± SEM of three ind. experiments, unpaired t-test). Immunofluorescence microscopy (two independent repeats) on RDEB-SCC1MIR10B−/− bulk culture compared to parental RDEB-SCC1. f Immunostaining of DIAPH2 (green) and DAPI (blue) on HC-skin compared to HC-cSCC, RDEB-skin and RDEB-cSCC FFPE tissue sections. Scale bars = 50 μm (c, e, f), and 1000 μm for H&E stainings