Therapeutic resistance to ionizing radiation (IR) limits the effectiveness of radiotherapy, which is the only potentially curative nonsurgical approach for most tumors, including non-small cell lung cancer (NSCLC). IR kills tumor cells mainly by inducing DNA double-strand breaks (DSBs) however, efficient DSB repair in tumor cells frequently prevents successful treatment. The possibility to inhibit 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 radiosensitization. MiRNAs negatively regulate gene expression by repressing translation or inducing degradation of target mRNAs. The goal of this study is to identify miRNAs targeting genes of homologous recombination (HR) and non-homologous end joining (NHEJ) to affect DSB repair of NSCLC cells. Our strategy consists in the delivery into cancer cells of synthetic miRNAs targeting DSB repair genes to decrease the expression level of related repair proteins making the cells more sensitive to IR. To this aim we constructed luciferase vectors containing the 3’UTR of genes involved in HR and NHEJ (classical and alternative) and determined whether they are real targets of selected miRNAs by functional validation with luciferase assay, site-directed mutagenesis and western blot. The in vivo effectiveness of miRNA treatments has been evaluated through experiments of clonogenic survival, cell cycle progression and DSB repair efficiency. The contribution of HR and NHEJ repair is under investigation to elucidate the role of selected miRNA-mRNA interaction in DSB repair of NSCLC cells.

Role of miRNAs targeting genes of DNA double-strand break repair in radioresistant non-small lung cancer cells

Piotto C;PETTENUZZO, MARISA;Barbieri V;Biscontin A;Mognato M
2017

Abstract

Therapeutic resistance to ionizing radiation (IR) limits the effectiveness of radiotherapy, which is the only potentially curative nonsurgical approach for most tumors, including non-small cell lung cancer (NSCLC). IR kills tumor cells mainly by inducing DNA double-strand breaks (DSBs) however, efficient DSB repair in tumor cells frequently prevents successful treatment. The possibility to inhibit 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 radiosensitization. MiRNAs negatively regulate gene expression by repressing translation or inducing degradation of target mRNAs. The goal of this study is to identify miRNAs targeting genes of homologous recombination (HR) and non-homologous end joining (NHEJ) to affect DSB repair of NSCLC cells. Our strategy consists in the delivery into cancer cells of synthetic miRNAs targeting DSB repair genes to decrease the expression level of related repair proteins making the cells more sensitive to IR. To this aim we constructed luciferase vectors containing the 3’UTR of genes involved in HR and NHEJ (classical and alternative) and determined whether they are real targets of selected miRNAs by functional validation with luciferase assay, site-directed mutagenesis and western blot. The in vivo effectiveness of miRNA treatments has been evaluated through experiments of clonogenic survival, cell cycle progression and DSB repair efficiency. The contribution of HR and NHEJ repair is under investigation to elucidate the role of selected miRNA-mRNA interaction in DSB repair of NSCLC cells.
Defects in the DNA damage response as targets for personalized therapeutic approaches; DNA damage, epigenetics and DNA repair
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3255976
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