e13515 Background: The DNA damage repair (DDR) pathways are an essential cell survival mechanism to preserve genomic integrity. In tumor cells, DDR pathways have a huge impact in cell survival, response to therapy and thus prognosis. Our objective is to investigate whether microRNAs (miRs) can influence specific DNA repair pathways. For our studies we focussed on double-strand break (DSB) repair by homologous - (HR) or non-homologous recombination (NHEJ). DSBs are the main lethal lesions induced by ionizing radiation. Methods: A retroviral miR expression library of ~500 miRs was transduced into a HR reporter cell line. For less abundant miRs found in this analysis in silico analysis was performed to detect potential targets. Regulation of HR protein RAD51 by miR-124 could be shown by luciferase assays and immunoblotting. Overexpression of miR-124 in glioblastoma cell line U87 was done by lentiviral constructs. Effect of miR-124 overexpression was revealed by colony – and competetive growth assays. Results: In an unbiased screen we found that miR-124 is less abundantly expressed in a reporter cell population sufficient for HR, suggesting a negative role for these miRs in DSB repair via HR. Reporter assays revealed a negative regulation of RAD51, a key protein of HR pathway, by miR-124. This regulation was specific to a conserved binding motif at the 3` UTR of RAD51. This regulation could also be detected in vivo whereas overexpression of miR-124 leads to downregulation of RAD51 protein levels. Functional experiments in glioblastoma cell line (U87), that express hardly no miR-124, revealed increased sensitivity to ionizing radiation if miR-124 is (re)-expressed. Conclusions: In this work we uncover miRs influencing the cellular DNA-repair machinery. We show that miR-124 influences the repair of DSBs by HR. This effect is specific and mediated by the regulation of RAD51 protein level, an essential component of the HR pathway. Re-expression of miR-124 sensitizes glioblastoma cell line (U87) to radiation in functional experiments. We propose that (re)-expressing miR-124 in glioblastomas could be a new treatment strategy to sensitize this tumors to radiation therapy.
Expression of microRNAs associated with apoptosis in neurospheres and adhered cells in glioblastoma cell line culture treated with ionizing radiation and temozolomida (803.14)
