The clinical samples were collected from local hospital with informed consent

ll-cell lung cancer cell line. Eight of the 14 proteins predicted to be repressed by the radiation up-regulation of miR-525-3p were confirmed by luciferase reporter assays to be direct targets. In the absence of miR-525-3p these 8 reporter constructs were all overexpressed in irradiated cells confirming that the miR-525-3p:: target interactions occur under physiological conditions. miRNA target interaction is mainly based on a stringent base pairing between the miRNA seed sequence and the target mRNA. Three of the direct targets in this study contained such stringent seed sequence matches. The remaining five direct targets showed only weak predicted seed sequence interactions. Such experimentally verified targets with poor seed sequences matches are not unusual. It is suggested that additional 3- pairing and pairing in centered regions of miRNAs could compensate for weaker seed sequence binding. Also, a recently discovered alternative binding mechanism involving a multistep binding process with induced conformational changes in the miRNA:: mRNA duplex may support binding between miRNA and targets with poor seed sequence matches. Four of the eight direct miR-525-3p targets, ARRB1, hnRNPK, HSPA9 and TXN1 have functions in the cellular stress response. As none of these proteins were significantly increased in miR-525-3p competent cells in response to irradiation we can assume that increases in their expression levels are suppressed during the radiation response by the action of the increase in miR-525-3p. It is possible that lowlevel changes in their regulation may occur below the detection limit of our proteomic analysis. Individual analysis of the changes of these four targets after irradiation confirmed that ARRB1 and TXN1 act as negative regulators of 23103164 survival. Cell purchase c-Met inhibitor 2 survival increased after irradiation when these proteins were knocked down by siRNA. In contrast, HSPA9 has a direct pro-survival function, with HSPA9-depleted cells being more radiosensitive than controls. Integrating these results with the overall effect of miR-525-3p on radiation sensitivity we suggest that the up-regulation of miR-525-3p acts to fine tune the balance between both, the negative and the positive regulators of survival. 10 miR-525-3p Mediated Survival after Irradiation doi: 10.1371/journal.pone.0077484.g006 11 miR-525-3p Mediated Survival after Irradiation The repressed protein ARRB1 indirectly regulates transcription factors involved in DNA damage processing and apoptosis in chronic stress responses through binding to 18201139 regulators such as IB and MDM2. Suppression of ARRB1 by RNA interference increases NF-B activity in HeLa cells and, conversely, its overexpression reduces NF-B activity. Further, ARRB1 suppresses p53 levels leading to an accumulation of unrepaired DNA damage. The radiation-induced increase of ARRB1 in cells with repressed miR-525-3p may serve to reduce NF-B activity leading to increased radiosensitivity and apoptosis. TXN1 is a cellular redox enzyme that controls the activation of a number of transcription factors participating in the radiation response. Byun et al. have shown that increased TXN1 expression is associated with elevated radiation sensitivity through increased apoptosis and senescence. We propose similar consequences for the radiation-induced up-regulation of TXN1 in miR-525-3p blocked cells. Indeed, the siRNAmediated knockdown of TXN1 led to increased survival and reduced apoptosis after irradiation. HSPA9 has been shown to in

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