The cellular response to radiation induced DNA damage determines the outcome of radiotherapy. The skin is a normal tissue that is always affected in radiotherapy, and exhibits both early and late effects that can be dose-limiting. However, the molecular mechanisms behind the side effects are still poorly understood The DNA-damage response pathways consist of a network of genes activated to repair DNA insult and promote cell survival. MicroRNAs (miRNAs) are short non-protein-coding RNAs that regulate ~30% of all gene expressions. Many miRNAs are tumor suppressors and activated by DNA damage. The role of miRNAs in the response to radiation induced DNA damage could be crucial and contribute to the different side effects seen in normal tissues.
The objective of the present clinical study is to elucidate the potential impact of miRNA regulation of DNA damage response in normal skin to radiotherapy given with daily fractions of 2 Gy over 5 weeks. We have focused on miR-34a, miR-16 and MiR-203, known to be upregulated by p53 upon various types of genotoxic exposure.
A total of 141 punch skin biopsies from 15 breast cancer patients were collected before, during and after radiotherapy. The skin was microdissected into epidermis and dermis. Micro-RNA levels were determined for miR-34a, miR-16 and miR-203 by RT-QPCR. Associated protein levels of p21, Bcl-2 and p63 were determined by immunohistochemistry.
miR-34a was induced in a very similar pattern as p21 during the radiotherapy course. Also the decline rate of the miR-34a expression following the end of radiotherapy was equal to that of p21. The relative expression levels of miR-34a and p21 increased both in epidermis and dermis, to the same degree for the two different tissues during the treatment period (p< 0.002). After completion of treatment the miR-34a and p21 levels declined faster in the epidermis compared to dermis (p<0.003). The expression levels in dermis remained elevated for weeks for both miR-34a and p21. No change could be observed for miR-16 neither in epidermis or dermis, as well as in the expression of Bcl-2. Notably, no induced changes in miR-203 and p63 were detectable in epidermis. Unexpectedly, there was a significant reduction of miR-203 in the dermis.
This study is the first to show a significant up-regulation of miR-34a in various normal cell populations to the DNA damage inflicted by radiotherapy. The miR-34a induction is of equal degree in epidermis and dermis and mirrors that of p21, a well-known target protein of p53. Secondly, in contrast to the successive decline of p21 and miR-34a in epidermal keratinocytes after completion of treatment, retention lasting for weeks is characteristic for fibroblasts and endothelial cells in dermis. This diverging molecular resolution of the DNA damage reveals early mechanistic differences in the pathological processes behind acute reversible effects of epidermis and late progressive changes of dermis. Our findings highlight that the accessibility makes the skin to a unique clinical model to quantify markers of DNA damage response to repeated DNA insult as it is delivered in radiotherapy.