Radiotherapy represents one of the leading approaches in cancer therapy, however, therapeutic resistance to ionizing radiation (IR) is the primary factor that limits the effectiveness of radiotherapy. IR kills tumor cells mainly by inducing DNA double-strand breaks (DSBs) causing reproductive cell death; however, the efficient repair of DSBs in tumor cells frequently prevents successful treatment. The possibility to affect DNA DSB repair may represent an efficient way to sensitize human cells to IR induced killing. To this purpose, microRNAs (miRNAs) could have an important role in tumor radio-sensitivity. MiRNAs are endogenous non-coding RNA molecules that suppress gene expression by binding to the complementary sequence in the 3’-untranslated region (UTR) of target transcripts. Therefore, the delivery into cells of synthetic miRNAs that mimic miRNAs targeting genes of DNA DSB repair can perturb the process making the cells more sensitive to IR. We constructed luciferase vectors containing the 3’UTR of RAD51, DNA-PKcs and LIG1 genes, involved respectively in homologous recombination (HR), in classical non-homologous end joining (c-NHEJ) and in alternative non-homologous end joining (a-NHEJ) and by luciferase assays we selected and validated miRNAs targeting such genes. Then, we over-expressed these miRNAs in radio-resistant cancer cells before exposing the cells to y-irradiation. Our results clearly show that clonogenic cell survival after irradiation is significantly decreased in miRNA-transfected cells than in mock-transfected control cells.

Sensitizing radio-resistant cancer cells to ionizing radiation through miRNA-based treatments.

PIOTTO, CELESTE;BISCONTIN, ALBERTO;MOGNATO, MADDALENA
2016

Abstract

Radiotherapy represents one of the leading approaches in cancer therapy, however, therapeutic resistance to ionizing radiation (IR) is the primary factor that limits the effectiveness of radiotherapy. IR kills tumor cells mainly by inducing DNA double-strand breaks (DSBs) causing reproductive cell death; however, the efficient repair of DSBs in tumor cells frequently prevents successful treatment. The possibility to affect DNA DSB repair may represent an efficient way to sensitize human cells to IR induced killing. To this purpose, microRNAs (miRNAs) could have an important role in tumor radio-sensitivity. MiRNAs are endogenous non-coding RNA molecules that suppress gene expression by binding to the complementary sequence in the 3’-untranslated region (UTR) of target transcripts. Therefore, the delivery into cells of synthetic miRNAs that mimic miRNAs targeting genes of DNA DSB repair can perturb the process making the cells more sensitive to IR. We constructed luciferase vectors containing the 3’UTR of RAD51, DNA-PKcs and LIG1 genes, involved respectively in homologous recombination (HR), in classical non-homologous end joining (c-NHEJ) and in alternative non-homologous end joining (a-NHEJ) and by luciferase assays we selected and validated miRNAs targeting such genes. Then, we over-expressed these miRNAs in radio-resistant cancer cells before exposing the cells to y-irradiation. Our results clearly show that clonogenic cell survival after irradiation is significantly decreased in miRNA-transfected cells than in mock-transfected control cells.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3216501
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