MiR-96 downregulates REV1 and RAD51 to promote cellular sensitivity to cisplatin and PARP inhibition

Abstract

Cell survival after DNA damage relies on DNA repair, the abrogation of which causes genomic instability. The DNA repair protein RAD51 and the trans-lesion synthesis DNA polymerase REV1 are required for resistance to DNA interstrand cross-linking agents such as cisplatin. In this study, we show that overexpression of miR-96 in human cancer cells reduces the levels of RAD51 and REV1 and impacts the cellular response to agents that cause DNA damage. MiR-96 directly targeted the coding region of RAD51 and the 3'-untranslated region of REV1. Overexpression of miR-96 decreased the efficiency of homologous recombination and enhanced sensitivity to the PARP inhibitor AZD2281 in vitro and to cisplatin both in vitro and in vivo. Taken together, our findings indicate that miR-96 regulates DNA repair and chemosensitivity by repressing RAD51 and REV1. As a therapeutic candidate, miR-96 may improve chemotherapeutic efficacy by increasing the sensitivity of cancer cells to DNA damage.

Figure 1. MiR-96 inhibits DNA damage-induced RAD51 foci formation

(A–C) U2OS cells were transfected with negative control (miR-neg) or miR-96 mimics (10 nM), treated with ionizing radiation (IR) (10 Gy) and then fixed for immunofluorescent staining of RAD51, RPA2, FANCD2 or BRCA1 six hours after IR. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). The representative images of RAD51 immunostaining were shown in (A). Quantitation of relative percentage of cells with at least 10 foci (normalized to miR-neg-transfected cells) and average number of foci per cell were shown in (B) and (C), respectively. (Scale bar = 20 μm) (mean ± SD, n=3). (D) U2OS cells were transfected with miR-neg, miR-96 or miR-16, a known cell cycle regulator (49). After 48 hours, cells were processed for FACS analysis of cell cycle profile. (E–F) U2OS cells were transfected with miR-neg or miR-96 and cells were harvested 48 hours following transfection for western blot analysis (E) or real-time RT-PCR.

Figure 2. MiR-96 inhibits homologous recombination and sensitizes U2OS cells to cisplatin and AZD2281

(A–B) U2OS DR-GFP cells were sequentially transfected with miRNA mimics or siRNAs and HA-tagged pBASceI plasmid. Cells were incubated for 48 hours and then processed for FACS analysis of GFP-positive cells in HA-positive population (mean ± SD, n=5). *** P < 0.001 (C–D) U2OS cells were transfected with miRNA mimics and reseeded 48 hours after transfection for cisplatin (C), and AZD2281 (D) sensitivity assays (mean ± SEM, n=3). * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 3. Downregulation of RAD51 is critical for miR-96-mediated AZD2281 sensitivity, but not for cisplatin sensitivity

(A–C) U2OS cells were infected with retrovirus expressing control (ctrl) or RAD51. After 72 hours, cells were reseeded and transfected with miR-neg or miR-96 mimics. After another 48 hours, cells were either reseeded for AZD2281 (B) and cisplatin (C) sensitivity assays (mean ± SEM, n=3) or harvested for western blot analysis (A). (D–F) U2OS cells were infected with retrovirus producing shRNAs against control (ctrl) or BRCA1. After 72 hours, cells were reseeded and transfected with miR-neg or miR-96 mimics. After another 48 hours, cells were plated for AZD2281 (E) and cisplatin (F) sensitivity assays (mean ± SEM, n=3) or harvested for western blot analysis (D).

Figure 4. Downregulation of REV1 is critical for miR-96-mediated cisplatin sensitivity

(A–B) U2OS cells were transfected with miRNA mimics or infected with retrovirus producing shRNAs as indicated. After 72 hours, cells were harvested for western blot analysis (A) and real time RT-PCR (B). (C) U2OS cells expressing control or REV1 shRNAs were plated for cisplatin sensitivity assay (mean ± SEM, n=3). (D–E) U2OS cells were infected with lentivirus expressing control (ctrl) or RAD51 and REV1. After 72 hours, cells were reseeded and transfected with miR-neg or miR-96 mimics. After another 48 hours, cells were plated for cisplatin sensitivity assays (E) (mean ± SEM, n=3) or harvested for western blot analysis (D). * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 5. RAD51 and REV1 are direct targets of miR-96

(A, B) Putative miR-96 binding sites (wildtype and mutants) in the transcripts of RAD51 or REV1. (C) Wildtype or mutant RAD51 3′-UTR, RAD51 MRE2 or REV1 3′-UTR were cloned into the pGL3 vector, as 3′ fusions to the luciferase gene. U2OS were co-transfected with the indicated miRNA mimics and luciferase vectors. Luciferase activity was assayed 48 h later and normalized to that of negative control-transfected cells (mean ± SD, n=4). (D) U2OS cells were co-transfected with plasmids and miRNA inhibitors (25 nM) as indicated. Cells were lysed for luciferase assay 48 h later. (E) HCT116 cells were transfected with miRNA mimics or inhibitors. Cells were harvested for western blot 72 h after transfection. * P < 0.05, ** P < 0.01, *** P < 0.001

Figure 6. MiR-96 enhances cisplatin sensitivity in vivo

(A) Overexpression of miR-96 in MDA-MB-231 cells by lentiviral delivery of pLemiR-96. (B) Reduction of REV1 and RAD51 expression in MDA-MB-231 cells stably expressing miR-96. (C) Overexpression of miR-96 mildly sensitizes MDA-MB-231 cells to cisplatin during the 5-day in vitro survival assay (mean ± SEM, n=3). (D) MDA-MB-231 cells stably expressing miR-96 were injected into mice subcutaneously. After the tumor was established, mice were i.p. injected with 20 mg/kg cisplatin followed by monitoring of tumor size for 3 weeks (mean ± SEM, n=9). * P < 0.05, ** P < 0.01