Figure 1.
Expression of miR-525-3p is up-regulated after ionizing radiation and modulation of the miR-525-3p expression effects cell survival.
(A) left miR-525-3p expression was examined 0, 2, 4, 8, 12, 24 and 48 h after 2.5 Gy IR in the endothelial cell line EA.hy926 by quantitative real time PCR. right modulation of miR-525-3p results in a change in endothelial cell proliferation after IR. Endothelial cells were transfected with pre-miR-525-3p, miR-525-3p-inhibitor or scrambled control RNA, reseeded and the cell proliferation assay was performed 5d after IR. (B) HeLa cells, (C) RPE cells, (D) U2-OS cells. The mean ± s.e.m. of three independent experiments is shown. * mark significant differences between samples harvested at the 0 h timepoint compared with the indicated time point (* p<0.05, ** p< 0.01).
Figure 2.
2D-DIGE gel showing the proteome of EA.hy926 cells at pH range of 3-11.
Differentially regulated spots in control transfected cells (0 Gy) versus anti-miR-525-3p treated cells (2.5 Gy) are indicated with corresponding spot numbers.
Figure 3.
Verification of direct miR-525-3p targets using luciferase assays.
(A) Relative luciferase activities after a co-transfection of luciferase constructs and control miRNA or pre-miR-525-3p in EA.hy926 cells. The mean ± s.e.m. of three independent experiments is shown. * indicate significant differences to pmiRGlo transfected cells (* p<0.05, ** p< 0.01). (B) Relative luciferase activities after co-transfection of luciferase constructs and anti-miR-525-3p or control miRNA followed by an irradiation with 2.5 Gy. The Firefly luciferase values were normalized for transfection with Renilla luciferase activity. Relative luciferase activities represent the ratio between normalized luciferase activities of pre-miR-525-3p and control miRNA transfected cells. Grey dashed bars represent sequences with perfect seed sequence matches to miR-525-3p. The mean ± s.e.m. of three independent experiments is shown. * indicate significant differences between control and anti-miR-525-3p transfected cells (* p<0.05, ** p< 0.01). (C) Complementarity of miR-525-3p sequence to the three target genes bearing perfect seed matches. The seed sequence is shown in red. Vertical lines indicate identity between miRNA sequence and corresponding gene sequence.
Figure 4.
Ingenuity pathway analysis of proteins deregulated 12 h after irradiation in the absence of miR-525-3p.
(A) IPA of direct and indirect miR-525-3p target proteins. The most significant network ”Cell Death and Survival, Free Radical Scavenging, Cancer” (score 26) is shown. (B) IPA of direct miR-525-3p target proteins. The most significant network “Cell Death and Survival, Organismal Injury and Abnormalities, Respiratory Disease” (score 14) is shown. Molecules in grey represent miR-525-3p target proteins. direct interaction, ------ indirect interaction.
Figure 5.
Immunoblot analysis of control and anti-miR-525-3p transfected EA.hy whole cell extracts (12 h after irradiation).
(A) Representative images of the blots. (B) Fold differences between irradiated and non-irradiated samples normalized to PCNA. * indicate significant differences between irradiated and non-irradiated samples (p< 0.05). The mean ± s.e.m. of three independent experiments is shown.
Figure 6.
Radiation response after depletion of ARRB1, TXN1, hnRNP
K and HSPA9. (A) siRNA-mediated knockdown of ARRB1 and TXN1. EA.hy926 cells were transfected with siARRB1 or with an unspecific control (siControl). ARRB1 and TXN1 were quantified 24 h after transfection by western blot. (B) siRNA-mediated knockdown of hnRNP K and HSPA9. (C) Proliferation activity after IR in ARRB1 and TXN1 knockdown cells. Depletion of ARRB1 and TXN1 results in increased radiation resistance after irradiation up to 7.5 Gy. Endothelial EAhy926 cells were transfected with siARRB1, siTXN1 or scrambled control RNA (siControl), reseeded and the cell proliferation assay was performed 5d after ionizing radiation. The mean ± s.e.m. of two independent experiments is shown. (D) Proliferation activity after IR in hnRNP K and HSPA9 knockdown cells. Depletion of hnRNP K did not change the proliferative activity and depletion of HSPA9 led to decreased proliferative activity. The mean ± s.e.m. of two independent experiments is shown. (E) Apoptosis induction in knockdown cells after IR. Apoptosis induction was quantified by sub-G1 analysis 48 h after IR. Depletion of ARRB1 and TXN1 led to decreased apoptosis, while depletion of HSPA9 increased apoptosis. * indicate significant difference to the respective siControl transfected cells (** p < 0.05, * p < 0.01). The mean ± s.e.m. of three independent experiments is shown.