2012a;125:3529C3534. via promoter hypermethylation occurs in approximately 10-20% of EOCs. Due to the underlying defect in DNA repair via homologous recombination (HR), patients with mutations (Fong et al., 2009). However, a substantial portion of these patients do not respond or eventually develop resistance to these brokers suggesting that and acquired platinum and PARPi resistance is a significant clinical problem in HR-defective EOCs. The most common mechanism of resistance to these brokers in causes a significant decrease in the level of genomic instability (chromosomal aberrations) induced by olaparib treatment (Fig. 2C). To address the mechanism by which miR-622 promotes genome integrity in mutant cells, we tested whether its expression could cause an increase in irradiation-induced Rad51 foci, a measure of the HR-pathway. We found that expression of miR-622 in UWB1.289 cells caused a statistically significant increase in Rad51 foci (Fig. 2D). Importantly, none of these effects are due to alterations in the cell cycle caused by the miR-622 mimics (Supp Fig. 2A). Open in a separate window Physique 2 Impact of miR-622 on genome stability and NHEJ repair pathways(A, B) Measurement of Ned 19 C-NHEJ (A) or A-NHEJ (B) mediated repair of I-SceI induced site specific DSBs. Cells transporting a single copy of the recombination substrate with two tandem I-SceI sites were transfected with control mimic, miR-622 mimic, Ku70 siRNA or Ligase4 siRNA before transfection with I-SceI or control vector. In 48 hrs, GFP positive cells were Ned 19 analyzed by circulation cytometry. (C) Analysis of genomic instability in metaphases. BRCA1?/? MEF cells were transfected with control miRNA mimic or miR-622, treated with 100nM PARP inhibitor, and measured for abnormal chromosomes in metaphase. (n50 metaphases). (D) Analysis of HR-mediated repair by RAD51 focus formation. Ned 19 UWB1.289 cells were transfected with control miRNA mimic or miR-622, stained for RAD51 (green), H2AX (red) and 4,6-diamidino-2-phenylindole (DAPI) (blue) 6 hrs after exposure to 10Gy IR. The images were captured by fluorescence microscopy and RAD51 focus-positive cells (with 20 foci) were quantified by comparing 100 cells miR-622 regulates expression of the Ku complex To investigate the mechanism by which miR-622 influences NHEJ and impacts PARP inhibitor sensitivity we used a candidate-based approach whereby all genes implicated KRAS in NHEJ were screened for miRNA acknowledgement elements (MREs) of miR-622 using the PITA algorithm. This algorithm is unique in allowing G:U wobbles or seed mismatches, and identifies base pairing beyond the 5’end of the miRNA, predicts the sites not restricted to the 3’UTR of mRNA and identifies non-canonical MREs for specific miRNA/mRNA combinations(Lal et al., 2009). By using this algorithm, miR-622 was predicted to target the transcripts of 53BP1, Ku70, Ku80, APTX and APLF (Supp Fig. 3). We assessed the impact of over-expressing miR-622 in UWB1.289 cells around the mRNA level of these genes and observed a significant reduction in the transcripts of 53BP1, Ku70 and Ku80 (Fig. 3A). Subsequently, we decided the impact of these miRNAs around the protein level of their putative targets. Over-expressing miR-622 reduces the protein levels of Ku70 and Ku80 in UWB1.289 cells. The basal expression of the Ku proteins is lower in MEFs, and the impact of miR-622 Ned 19 on Ku70 and Ku80 in is usually even more pronounced (Fig. 3B). On the contrary, there was no detectable impact of miR-622 on 53BP1 in the UWB1.289 cells. To test for association of miR-622 with the Ku70 and Ku80 transcripts we captured miRNA-mRNA complexes using streptavidin-coated beads from cells transfected with biotinylated forms of the miRNA mimics (Lal et al., 2011; Orom and Lund, 2007). The amount of Ku70, Ku80 and 53BP1 transcripts was measured in the pull-downs, and the enrichment was assessed relative to pull-down with biotinylated control mimic and also with GAPDH. Consistent with our previous results, miR-622 selectively pulled-down Ku70 and Ku80 transcripts but not the 53BP1 transcript (Fig. 3C). To Ned 19 verify further that Ku70 and Ku80 are targets of miR-622 and confirm that the conversation is mediated by the predicted MREs we used luciferase reporter assays. The predicted MREs (Fig. 3D) were cloned in the 3’UTR of the luciferase gene, and expression monitored in cells.