Identification and validation of microRNAs targeting genes of DNA-Damage Response in human lymphocytes irradiated with γ-rays

Ionizing radiation (IR) used in radiotherapy to treat cancer cells is a source of DNA damage, among which double-strand breaks (DSBs) represent the most deleterious lesions. The major problems encountered when treating cancer patients with radiotherapy are toxic side effects and lack of efficacy owing to radioresistance. Many human tumors have a strong DNA repair capacity and so radiation therapy has low efficacy against cancer cells displaying radioresistant phenotypes. Therefore, the possibility to inhibit DNA repair pathways, and in particular DSB repair, may represent an efficient way to sensitize human cells to IR induced killing. In presence of DSBs eukaryotic cells activate a highly regulated system pathway-the DNA-Damage Response (DDR)-which encompasses damage sensors, mediators, signal transducers and effectors of cell cycle progression/arrest, DNA repair and apoptosis. The expression of DDR genes is modulated by microRNAs (miRNAs), small non-coding RNAs that act as post-transcriptional regulators of gene expression by direct association with target transcripts. The aim of our study is to assess the potential applicability of miRNA delivery in combination with ionizing radiation to increase the cytotoxic effects of IR in cancer cells. Indeed, the delivery into cancer cells of synthetic miRNAs that mimic endogenous miRNAs targeting genes of DDR pathway should perturb such process, making cells more sensitive to IR. To this purpose we are identifying and validating miRNAs targeting genes of DNA repair in human peripheral blood lymphocytes (PBLs) irradiated with γ-rays. We identified miRNA expression changes at two different times after irradiation (4 and 24h), with two different doses of radiation (0.2 and 2Gy). To improve the detection of target genes of radio-responsive miRNAs we analyzed mRNA expression profiles in irradiated PBLs and we integrated the transcriptome and microRNome to identify miRNAs modulating the expression of DDR genes. Our results show that few miRNA species are candidate to be essential regulators of genes of DDR, in particular, miRNAs targeting ATM, BAX, STAT5A, and genes of DNA repair DDB2, XPC, GADD45A, FANCF, LIG1. The in vitro validation with quantitative real-time PCR (qRT-PCR) and luciferase reporter assay confirmed several of the miRNA-mRNA interactions identified.