Page 30 - SDIR5 Abstract book 21 12 2021.

RADIOBIOLOGY

Short talks

Gene expression kinetics and pathway analysis of skin fibroblasts irradiated in vitro

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Sami Ahmad , MacKay Alan , Herskind Carsten 1
1 Cellular and Molecular Radiation Oncology Lab, Department of Radiation Oncology, Universitätsmedizin
Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
2 The Institute of Cancer Research, Division of Molecular Pathology, London, UK

Background: Fibroblasts are considered an important factor in the development of radiation-induced
subcutaneous fibrosis. The aim was to validate a previous gene expression study [1] and extend the kinetics
to 1-6 days after irradiation. Material and Methods: RNA was isolated from the early-passage skin fibroblast
strains (GS3, GS4, GS5), 1-6 days after X-irradiation with 4Gy (6MV) and analyzed on Breakthrough 20k
human expression microarrays. Biostatistical and bioinformatical analyses were performed with R and JMP
Genomics statistical software. Validation of selected genes and timepoints was performed in independent
experiments with quantitative real-time PCR (qPCR) and Western blotting. Results: Principal and variant
component analysis of the transcriptome pattern showed 42.2% dependence on time-point while 25.7%
was influenced by the biological background and 32.1% by residual factors. After adjustment for biological
background, Hierarchical Clustering analysis identified two subgroups of irradiated samples: early (1-3 days)
and late (4-6 days) after irradiation. After Two-Way ANOVA analysis between three groups (non-irradiated,
early and late), 3196 statistically significant differentially expressed genes (DEGs) were identified. 991 genes
were early DEGs and 2252 genes were late DEGs relative to unirradiated controls, while 2013 DEGs were
differentially regulated between the late and early timepoints. Pathway analysis was performed for each
consecutive time point. Radiation-induced downregulation of cell-cycle regulatory processes and
upregulation of ECM regulation processes confirmed the previous findings [1]. Conclusion: The present
study validates and extends findings from the previous study using a different microarray platform [1]. Ref.
[1]: Herskind et al., Front Cell Dev Biol 9:539893 (2021).
Keywords: fibroblasts, gene expression, pathway analysis, radiotherapy, skin fibrosis

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