Functional validation of miRNAs targeting genes of DNA double-strand break repair to radiosensitize non-small lung cancer cells

Abstract DNA-Double strand breaks (DSBs) generated by radiation therapy represent the most efficient lesions to kill tumor cells, however, the inherent DSB repair efficiency of tumor cells can cause cellular radioresistance and impact on therapeutic outcome. Genes of DSB repair represent a target for cancer therapy since their down-regulation can impair the repair process making the cells more sensitive to radiation. In this study, we analyzed the combination of ionizing radiation (IR) along with microRNA-mediated targeting of genes involved in DSB repair to sensitize human non-small cell lung cancer (NSCLC) cells. MicroRNAs are natural occurring modulators of gene expression and therefore represent an attractive strategy to affect the expression of DSB repair genes. As possible IR-sensitizing targets genes we selected genes of homologous recombination (HR) and non-homologous end joining (NHEJ) pathway (i.e. RAD51, BRCA2, PRKDC, XRCC5, LIG1). We examined these genes to determine whether they may be real targets of selected miRNAs by functional and biological validation. The in vivo effectiveness of miRNA treatments has been examined in cells over-expressing miRNAs and treated with IR. Taken together, our results show that hsa-miR-96-5p and hsa-miR-874-3p can directly regulate the expression of target genes. When these miRNAs are combined with IR can decrease the survival of NSCLC cells to a higher extent than that exerted by radiation alone, and similarly to radiation combined with specific chemical inhibitors of HR and NHEJ repair pathway

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

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\u2019-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\u2019UTR 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

Immune Regulation of Tissue Repair and Regeneration via miRNAs—New Therapeutic Target

The importance of immunity in tissue repair and regeneration is now evident. Thus, promoting tissue healing through immune modulation is a growing and promising field. Targeting microRNAs (miRNAs) is an appealing option since they regulate immunity through post-transcriptional gene fine-tuning in immune cells. Indeed, miRNAs are involved in inflammation as well as in its resolution by controlling immune cell phenotypes and functions. In this review, we first discuss the immunoregulatory role of miRNAs during the restoration of tissue homeostasis after injury, focusing mainly on neutrophils, macrophages and T lymphocytes. As tissue examples, we present the immunoregulatory function of miRNAs during the repair and regeneration of the heart, skeletal muscles, skin and liver. Secondly, we discuss recent technological advances for designing therapeutic strategies which target miRNAs. Specifically, we highlight the possible use of miRNAs and anti-miRNAs for promoting tissue regeneration via modulation of the immune system