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Systematic screen identifies miRNAs that target RAD51 and RAD51D to enhance chemosensitivity - PubMed

Systematic screen identifies miRNAs that target RAD51 and RAD51D to enhance chemosensitivity

Jen-Wei Huang et al. Mol Cancer Res. 2013 Dec.

Abstract

Homologous recombination mediates error-free repair of DNA double-strand breaks (DSB). RAD51 is an essential protein for catalyzing homologous recombination and its recruitment to DSBs is mediated by many factors including RAD51, its paralogs, and breast/ovarian cancer susceptibility gene products BRCA1/2. Deregulation of these factors leads to impaired DNA repair, genomic instability, and cellular sensitivity to chemotherapeutics such as cisplatin and PARP inhibitors. microRNAs (miRNA) are short, noncoding RNAs that posttranscriptionally regulate gene expression; however, the contribution of miRNAs in the regulation of homologous recombination is not well understood. To address this, a library of human miRNA mimics was systematically screened to pinpoint several miRNAs that significantly reduce RAD51 foci formation in response to ionizing radiation in human osteosarcoma cells. Subsequent study focused on two of the strongest candidates, miR-103 and miR-107, as they are frequently deregulated in cancer. Consistent with the inhibition of RAD51 foci formation, miR-103 and miR-107 reduced homology-directed repair and sensitized cells to various DNA-damaging agents, including cisplatin and a PARP inhibitor. Mechanistic analyses revealed that both miR-103 and miR-107 directly target and regulate RAD51 and RAD51D, which is critical for miR-103/107-mediated chemosensitization. Furthermore, endogenous regulation of RAD51D by miR-103/107 was observed in several tumor subtypes. Taken together, these data show that miR-103 and miR-107 overexpression promotes genomic instability and may be used therapeutically to chemosensitize tumors.

Implications: These findings demonstrate a role for miR-103 and -107 in regulating DNA damage repair, thereby identifying new players in the progression of cancer and response to chemotherapy.

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Conflict of interest statement

Potential conflicts of interest: none.

Figures

Figure 1
Figure 1. MiR-103 and miR-107 negatively regulate DNA damage-induced RAD51 foci formation

A. Screening strategy for the identification of microRNAs that regulate IR-induced RAD51 foci formation in U2OS cells. B. The mean Z-scores of each microRNA from the screen are ranked (n=3). A table of image analyses for miR-103, miR-107 and siRAD51 control is presented. C. Mature sequences of miR-103 and miR-107 differ at a single nucleotide (*). Seed regions are underlined. D. U2OS cells transfected with indicated microRNAs or siRNAs (10 nM) were irradiated (10 Gy) 48 hours later, fixed 6 hours post-IR, and immunostained for RAD51. Nuclei were counterstained with DAPI. RAD51 foci-positive cells contained at least 5 nuclear foci (n=3, +/−SEM). Representative images are shown (scale bar = 20 μm).

Figure 2
Figure 2. MiR-103 and miR-107 impair HR and promote chemosensitivity to DNA-damaging agents

A. U2OS DR-GFP cells transfected with indicated microRNAs (10 nM) were transfected the next day with an I-SceI expression vector and harvested 48 hours later for flow cytometry. U2OS DR-GFP cells depleted of BRCA2 (10 nM) served as a control of impaired HR. Representative flow cytometry profiles with gated GFP-positive populations are shown (n=4, +/−SEM). HR repair efficiency is presented as a percentage of the miR-Neg or siControl transfected controls. B-D. U2OS cells transfected with indicated microRNAs (10 nM) were reseeded 48 hours later to assess for cell growth in response to cisplatin and AZD2281 (B), etoposide and camptothecin (C), and paclitaxel (D). Cell grow this expressed as a fraction of the untreated control (n=3, +/−SEM).

Figure 3
Figure 3. MiR-103 and miR-107 directly target RAD51 and RAD51D

A. HeLa, U2OS and PEO1 C4-2 cells transfected with indicated microRNAs (10 nM) were harvested 48 hours later for Western blotting. B. HeLa and U2OS cells transfected with indicated microRNAs (10 nM) were harvested 48 hours later for real-time PCR. C. Putative miR-103/107 binding sites in the 3′UTRs of RAD51 and RAD51D. Asterisks denote positions of substitutions made to generate mutant 3′UTR constructs. The seed sequences of miR-103/107 are underlined. D. U2OS cells co-transfected with indicated microRNAs (0.5 nM) and luciferase constructs (x-axis) were assayed for luciferase activity 48 hours later (n=8-9, +/−SEM) (Student's t-test: *P< 0.05, **P< 0.005).E. H1299 cells stably expressing NSC, miR-103 or miR-107 were harvested two days after transfection with indicated microRNA inhibitors (30 nM) for Western blotting. F. Quantitation of target expression in H1299 cells stably expressing NSC and transfected with indicated microRNA inhibitors, as in (E). RAD51 and RAD51D protein levels were normalized to actin and then expressed relative to PI-Neg-transfected cells (n=3, +/−SD).

Figure 4
Figure 4. RAD51 and RAD51D targeting is relevant to miR-103/107-mediated regulation of RAD51 foci formation and chemosensitivity

A. PEO1 C4-2 cells stably expressing the indicated cDNAs were transfected with miR-Neg or miR-107 (10 nM), irradiated (10 Gy) 48 hours later, fixed 6 hours post-IR, and immunostained with anti-RAD51. Nuclei were counterstained with DAPI. RAD51 foci-positive cells contain at least 5 nuclear foci (n=3-4, +/−SEM) (Student's t-test:*P< 0.05, **P< 0.01, #not significant).B-C. Indicated PEO1 C4-2 stable cell lines transfected with miR-Neg or miR-107 (10 nM) were (B) harvested 48 hours later for Western blotting and (C) reseeded 48 hours later to assess cell growth in response to cisplatin and AZD2281. Cell growth is expressed as a fraction of the untreated control (n=3-4, +/−SEM) (Student's t-test: *P< 0.05, **P< 0.005, #not significant). D. Our model showing that the regulation of HR and cellular resistance to DNA-damaging agents by miR-103/107 is mediated through the targeting of RAD51 and RAD51D.

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