Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways

Abstract

Radiotherapy for brain tumors induces neuronal DNA damage and may lead to neurodegeneration and cognitive deficits. We investigated the mechanisms of radiation-induced neuronal cell death and the role of miR-711 in the regulation of these pathways. We used in vitro and in vivo models of radiation-induced neuronal cell death. We showed that X-ray exposure in primary cortical neurons induced activation of p53-mediated mechanisms including intrinsic apoptotic pathways with sequential upregulation of BH3-only molecules, mitochondrial release of cytochrome c and AIF-1, as well as senescence pathways including upregulation of p21^WAF1/Cip1. These pathways of irradiation-induced neuronal apoptosis may involve miR-711-dependent downregulation of pro-survival genes Akt and Ang-1. Accordingly, we demonstrated that inhibition of miR-711 attenuated degradation of Akt and Ang-1 mRNAs and reduced intrinsic apoptosis after neuronal irradiation; likewise, administration of Ang-1 was neuroprotective. Importantly, irradiation also downregulated two novel miR-711 targets, DNA-repair genes Rad50 and Rad54l2, which may impair DNA damage responses, amplifying the stimulation of apoptotic and senescence pathways and contributing to neurodegeneration. Inhibition of miR-711 rescued Rad50 and Rad54l2 expression after neuronal irradiation, enhancing DNA repair and reducing p53-dependent apoptotic and senescence pathways. Significantly, we showed that brain irradiation in vivo persistently elevated miR-711, downregulated its targets, including pro-survival and DNA-repair molecules, and is associated with markers of neurodegeneration, not only across the cortex and hippocampus but also specifically in neurons isolated from the irradiated brain. Our data suggest that irradiation-induced miR-711 negatively modulates multiple pro-survival and DNA-repair mechanisms that converge to activate neuronal intrinsic apoptosis and senescence. Using miR-711 inhibitors to block the development of these regulated neurodegenerative pathways, thus increasing neuronal survival, may be an effective neuroprotective strategy.

Keywords: MOMP; Noxa; Puma; Rad54l2; microRNA (miR), Rad50; neuronal apoptosis; radiation.

Figure 1

Expression of anti-apoptotic and neuronal marker genes is downregulated, while expression of pro-apoptotic and pro-senescence genes is upregulated, in the cortex, hippocampus and purified neurons after brain irradiation. Experimental rationale and details are described in Experimental Setup. Tissues and neurons were collected at 30 min, 6 h, 24 h and 7 d after 10Gy whole-brain irradiation. Total RNA was used for qPCR analysis. qPCR quantification of (A) Akt1 mRNAs in cortex (F (4,25) = 40.18), hippocampus (F (4,25) = 18.50) and isolated neurons (F (4,20) = 15.48); (B) Ang-1 mRNAs in cortex (F (4,25) = 8.438), hippocampus (F(4,25) = 20.14) and isolated neurons (F(4,20) = 15.76); (C) p21 mRNAs in cortex (F(4,25) = 128.4), hippocampus (F(4,25) = 82.96) and isolated neurons (F(4,20) = 44.66); (D) Bim in hippocampus (F(4,25) = 44.84) and isolated neurons (F(4,20) = 11.61) and (E) Syn1s (F(4,25) = 19.18) mRNAs in hippocampus. n = 6/group for tissues, n = 6/group for isolated neurons, with 2 technical replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control animals non irradiated animals.

Figure 2

Expression of miR-711 is upregulated, while expression DNA-repair genes is downregulated, in the cortex, hippocampus and purified neurons after brain irradiation. Experimental rationale and details are described in Experimental Setup. Tissues and neurons were collected at 30 min, 6 h, 24 h and 7 d after 10Gy whole-brain irradiation. Total RNA was used for qPCR analysis. qPCR quantification of (A) miR-711 in cortex (F(4,25) = 24.46), hippocampus (F(4,25) = 34.3), and isolated neurons (F(4,20) = 20.88); (B) Rad50 mRNAs in cortex (F(4,25) = 41.65), hippocampus (F(4,25) = 23.99) and isolated neurons (F(4,20) = 10.74); (C) Rad54l2 mRNAs in cortex (F(4,25) = 7.513), hippocampus (F(4,25) = 35.05) and isolated neurons (F(4,20) = 17.2). n = 6/group for tissues, n = 6/group for isolated neurons, with 2 technical replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control animals non irradiated animals.

Figure 3

Ionizing radiation (IR) induces rapid and dose-dependent activation of DNA-damage and p53 pathways in primary cortical neurons. (A) Western blots for Ph-ATM(Ser 1981), γ-H2A.X, p53 and p21. (B) Ph-ATM (Ser1981) (F(6,14) = 246.4, p < 0.0001 for 30 min 2, 8 and 32Gy, compared to control; p = 0.0384 for 24 h 8Gy, p = 0.0483 for 24 h 32Gy, compared to control; p < 0.0001 for 24 h groups, compared to 30 min and for 30 min 8Gy, compared to 30 min 2Gy. (C) γ-H2A.X (F(6,14) = 47.02, p < 0.0001 for 30 min 8 and 32Gy compared, to control; p < 0.0001 for 30 min 8Gy compared to 30 min 2Gy). (D) γ-H2A.X normalized to total H2A.X (F 6,14) = 102.2, p < 0.0001 for 30 min 8 and 32Gy, compared to control; p < 0.0001 for 30 min 8Gy, compared to 30 min 2Gy; p = 0.045 for 30 min 32Gy, compared to 30 min 8Gy). (E) H2A.X normalized to β-actin. (F) Ph-p53 (F(6,14) = 351.5, p < 0.0001 for 30 min 2, 8 and 32Gy, and for 24 h 8 and 32Gy; p < 0.0001 for 8Gy compared to 2Gy and 32Gy, compared to 8Gy at 30 min; p = 0.0006 for 24 h 8Gy, compared to 24 h 2Gy; p < 0.0001 for 24 h 32Gy, compared to 24 h 8Gy; p < 0.0001 for 24 h 8Gy and 32Gy, compared to control). (G) Ph-p53 normalized to total p53 (F(6,14) = 114.5, p < 0.0001 for 30 min 2, 8 and 32Gy; p = 0.0007 for 24 h 8Gy, p < 0.0001 for 24 h 32Gy, compared to control; p = 0.0016 for 30 min 8Gy, compared to 30 min 2Gy, p < 0.0001 for 30 min 32Gy, compared to 30 min 8Gy; p = 0.0292 for 24 h 8Gy, compared to 24 h 2Gy). (H) p53 normalized to β-actin. (I) p21 (F(6,14) = 32.95, p = 0.0011 for 24 h 2Gy, p < 0.0001 for 24 h 8Gy and p = 0.0001 for 24 h 32Gy, compared to control). Data presented as fold change to non-irradiated control levels after normalization. n = 3/group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. (J) Neurons were collected 30 min and 24 h after 2, 8 and 32Gy irradiation. Total RNA was used for qPCR analysis. qPCR quantification of p21 mRNA. (F(6,14) = 17.7, p < 0.0001 for all 24 h time points, compared to control). Data presented as fold change to non-irradiated control group. n = 3/group; **** p < 0.0001 vs. control.

Figure 4

Select pro-apoptotic members of the Bcl-2 family are upregulated in irradiated primary cortical neurons. Post-ChIP qPCR to examine occupancy of p53 on Noxa promoter region (A). (T(4) = 2.08, p = 0.0001). Data presented as fold change to non-irradiated control group. n = 3/group. Significance assigned based on one-tailed t-test, *** p < 0.001 versus control Rat primary cortical neurons (RCNs). qPCR quantification of Noxa (B) (F(9,40) = 63.76, p < 0.0001 for 24 h 2Gy and for 6 h and 24 h 8 and 32Gy, compared to control), Puma (C) (F(9,40) = 56.47, p = 0.0051 for 6 h 2Gy, p = 0.0002 for 30 min 32Gy, and p < 0.0001 for 6 and 24 h 8Gy and 32Gy, compared to control), Bim (D) (F(9,40) = 65.12, p = 0.0044 for 6 h 2Gy, p < 0.0001 for all-time points treated with 8 and 32Gy except 30 min 8Gy, compared to control). Data presented as fold change to non-irradiated control group. n = 5 + /group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. Irradiation induces dose-dependent neuronal cell death and time- and dose-dependent activation of caspase-dependent apoptosis. Irradiation induces dose-dependent neuronal cell death, and time- and dose-dependent activation of caspase-dependent apoptosis in RCNs. Neurons were irradiated with 2, 8 and 32Gy. Twenty-four hours later, LDH release was measured (E) (F(3,36)= 7.43, p = 0.0175 for 2Gy, p < 0.0001 for 8 and 32Gy). Data expressed as a percentage of levels of non-irradiated control. n = 10/group, * p < 0.05, **** p < 0.0001 vs. control. Neurons were collected 30 min and 24 h after 2, 8 and 32Gy IR. Whole-cell lysates were separated by SDS-polyacrylamide gel and immunoblotted with antibodies against cleaved caspase-3, PARP, α-fodrin, PSD95 and β-actin (F). (G) Quantification of levels of cleaved caspase 3 (F(6,14) = 248.2, p < 0.0001 for 24 h at all doses, compared to control), PARP (F(6,14) = 79.08, p < 0.0001 for 24 h at 8 and 32Gy, compared to control), α-fodrin (120kDa) (F(6,14) = 553.4, p < 0.0001 for 24 h at all doses, compared to control) and PSD95 (H) (F(6,14) = 35.55, p = 0.0087 for 30 min 2Gy, p < 0.0001 at all other doses/times, except 30 min 32Gy, compared to control). n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001 vs. control.

Figure 5

Irradiation causes upregulation of miR-711 and downregulation of its target genes expression. (A) qPCR quantification of miR-711 (F(12,26) = 224.6, p = 0.0264 for 6 h 2Gy; p < 0.0001 for 3 h and 6 h 8Gy, p = 0.0016 for 24 h 8Gy; p < 0.0001 for 30 min, 3 h and 6 h 32Gy, compared to control), Akt mRNA (F(12,52) = 15.04; p = 0.0035 for 3 h 2Gy; p = 0.0264 for 30 min 8Gy; p < 0.0001 for 6 h and 24 h 8Gy and for 32Gy at all time points, compared to control) and Ang-1 mRNA (F(12,52) = 28.39; p = 0.0103 for 6 h 2Gy; p = 0.0042 for 30 min 8Gy, p < 0.0001 for 3 h and 6 h 8 and 32Gy; p = 0.0236 for 30 min 32Gy; p = 0.0085 for 24 h 32Gy, compared to control). n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control RCNs. (B) Neurons were collected 6 h after 8Gy irradiation and subjected to RIP with Ago2 antibodies; samples were used for qPCR analysis. qPCR quantification of miR-711 (T(4) = 5.078, p = 0.0035), Akt mRNA T(4) = 20.41, p < 0.0001) and Ang-1 mRNA (T(4) = 8.45, p < 0.0001). n = 3/group. Significance assigned by one-tailed t-test, ** p < 0.01, *** p < 0.001 vs. control RCNs. miR-711 inhibition attenuates IR-induced downregulation of Akt and Ang-1. (C) RCNs were transfected with miR-711 inhibitor and miR-ve inhibitor 1 h before exposure to 8Gy. qPCR quantification of Akt mRNA (F(10,43) = 51.96, p < 0.0001 at 1, 3 and 6 h after IR with miR-ve inhibitor, compared to non-irradiated control; for miR-711 inhibitor, compared to miR-ve control, p < 0.0001 at 30 min and 1 h, p = 0.0010 at 3 h, p = 0.0356 at 6). Ang-1 mRNA (F(10,43) = 19.56, p < 0.0001 at 3, 6 and 24 h after IR with miR-ve inhibitor, compared to non-irradiated control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 at 3 and 24 h, p = 0.0003 at 6 h). n = 4 for controls, n = 5/group for treatments, * p < 0.05, *** p< 0.001, vs. control; ^ p < 0.05, ^^^ p < 0.001, ^^^^ p < 0.0001 vs. corresponding 8Gy + miR-ve inhibitor. (D) RCNs were treated with recombinant Ang-1 to final concentrations 50, 100, 200 and 400 ng/mL, or with 200 ng/mL of recombinant Ang-1 and 6.25uM Akt inhibitor or 25nMWortmannin and exposed to 8Gy. LDH release was measured 24 h after irradiation (F(7,40) = 58.08, p < 0.0001 for IR alone vs. control; p < 0.0001 for 8Gy with 200 or 400 ng/mL Ang-1 vs. IR; p < 0.0001 for both inhibitor-containing groups vs. 8Gy with 200ng/mL Ang-1). n = 6/group, **** p < 0.0001 vs. control RCNs, ^^^^ p < 0.0001 vs. IR alone RCNs, &&&& p < 0.0001 vs. 8Gy with 200 ng/mL Ang-1. (E) qPCR quantification of mature miR-711 (F(10,22) = 95.15, p = 0.0035 at 1 h, p < 0.0001 at 3, 6 and 24 h after IR with miR-ve inhibitor, compared to control) and pri-mir-711 (F(10,25) = 54.32, p = 0.0056 at 1 h, p < 0.0001 at 3 and 6 h, p = 0.0300 at 24 h after IR with miR-ve inhibitor, compared to control). n = 3/group, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control. (F) Neurons were treated as describe above. Cells were collected in 3 h for RIP analysis for levels of miR-711 (F(2,6) = 112.8, p < 0.0001 for both treatments, compared to control), Akt1 mRNA (F(2,6) = 54.17, p = 0.0001 after IR with miR-ve inhibitor, compared to control, p = 0.0034 for miR-711 inhibitor, compared to miR-ve inhibitor) and Ang-1 mRNA (F(2,6) = 788.7, p < 0.0001 for all comparisons). n = 3/group. *** p < 0.001, **** p < 0.0001 vs. control RCNs; ^^ < 0.01, ^^^ p < 0.001, ^^^^ p < 0.0001 vs. corresponding 8Gy + miR-ve inhibitor group.

Figure 6

miR-711 inhibition attenuates IR-induced neuronal apoptosis. Western blot for Puma, cleaved caspase-3, PARP, α-fodrin and β-actin (A). (B) Quantification of levels of Puma (F(10,22) = 3.48, p = 0.0007 at 1 h, p < 0.0001 at 3 and 6 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0312 at 30 min, p < 0.0001at 6 h), cleaved caspase-3 (F(10,22) = 5.25, p < 0.0001 at 6 and 24 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0005 at 6, p < 0.0001 at 24 h), cleaved PARP (89kDa) (F(10,22) = 296.3, p = 0.0180 at 3, p < 0.0001 at 6, p = 0.0047 at 24 h after IR with miR-ve inhibitor, compared to non-irradiated control for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0225 at 3, p < 0.0001 at 6 h), α-fodrin (120kDa) (F(10,22) = 132.9, p < 0.0001 at 6 and 24 h after IR with miR-ve inhibitor, compared to control for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0068 at 6 h, p < 0.0001 at 24 h). n = 3/group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control; ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001, ^^^^ p < 0.0001 vs. corresponding 8Gy + miR-ve inhibitor group. qPCR quantification (C) of PUMA mRNA (F(10,43) = 351.7, p < 0.0001 at all-time points after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 for 3 h and 6 h), Noxa mRNA (F(10,43) = 389.8, p = 0.0254 at 1, p < 0.0001 at 3 and 6, p = 0.0470 at 24 h after IR with miR-ve inhibitor, compared to control for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 for 3 and 6 h) and Bim mRNA (F(10,43) = 85.04, p < 0.0001 at 3 and 6 h after IR with miR-ve inhibitor, compared to control for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 at 6 and 24 h). n = 4 for control, n = 5/group for all other groups, * p < 0.05, **** p < 0.0001 vs. control; ^^^^ p < 0.0001 vs. corresponding 8Gy + miR-ve inhibitor group. Cytosolic fractions were used for Western blot for AIF-1, cytochrome c and GAPDH (D). Levels of AIF-1(E)(F(2,9) = 294.1, p < 0.0001 for all comparisons) and Cytochrome c (F)(F(2,9) = 4.62, p < 0.0001 for non-irradiated control vs. miR-ve inhibitor group and for miR-ve inhibitor vs. miR-711 inhibitor group). n = 3/group, **** p < 0.0001 vs. control RCNs; ^^^^ p < 0.0001 vs. 8Gy + miR-ve inhibitor group. (G) LDH was measured 24 h and 48 h after irradiation)(F(5,30) = 131.3, p < 0.0001 at 24 h and 48 after IR with miR-ve inhibitor, compared to non-irradiated control for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0002 at 24 h, p < 0.0001 at 48 h; For 24 h vs. 48 h 8gy + miR-ve inhibitor, p < 0.0001; For 24 h vs. 48 h 8Gy + miR-711 inhibitor, p = 0.0045). n = 3/group. **** p < 0.0001 vs. control RCNs; ^^^ p < 0.001, ^^^^ p < 0.0001 vs. corresponding 8Gy + miR-ve inhibitor group; && p < 0.01, &&&& p < 0.0001 vs. equivalent treatment 24 h after 8Gy.

Figure 7

miR-711 inhibition attenuates IR-induced DNA damage markers, p53 activation, and neuronal apoptosis and senescence markers. Western blot for Ph-ATM(Ser1981), γ-H2A.X and H2A.X (A). (B) Ph-ATM(Ser1981) (F(10,22) = 173, p < 0.0001 at 30 min, 1 h, 3 h and 6 h after IR with miR-ve inhibitor, compared to control for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001at 1 h and 3 h, p = 0.0001 at 6 h), γH2A.X normalized to β-actin (F(10,22) = 281.7, p < 0.0001 for all time points except 24 h after IR with miR-ve inhibitor, compared to non-irradiated control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 for 1 h and 3 h, p = 0.0005 for 6 h), γH2A.X normalized to H2A.X (F(10,22) = 73.18, p < 0.0001 for all time points except 24 h after IR with miR-ve inhibitor, compared to control for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0017 at 1 h, p < 0.0001 at 3 h and 6 h). H2A.X (F(10,22) = 138.8, p < 0.0001 for 1 h and 3 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 for 1 h and 3 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 at 30 min, p = 0.0029 at 1 h). Western blots for Ph-ATR(Ser428), Ph-p53(Ser15), p53, p21 and β-actin (C). (D) Ph-ATR(Ser428)(F(8,18) = 108.4, p < 0.0001 at 30 min, 3 h and 6 h, p = 0.0017 at 24 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 at 3 h and 6 h), Ph-p53(Ser15) normalized to β-actin (F(8,18) = 302, p < 0.0001 at 3 h and 6 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 at 3 h and 6 h), Ph-p53(Ser15) normalized to p53(F(8,18) = 392.6, p < 0.0001 at 3 h and 6 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 at 3 h and 6 h), p21(F(8,18) = 45.58, p < 0.0001 at 3 h and 6 h, p = 0.0410 at 24 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0001 at 6 h). n = 3/group. qPCR quantification of p21 and p53 mRNAs (E). p21 mRNA (F(10,25) = 111, p = 0.0342 at 30 min, p < 0.0001 at 3 h and 6 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 at 3 h and 6 h), p53 mRNA (F(10,25) = 5.062, no significant changes). n = 3/group. * p < 0.05, **** p < 0.0001 vs. control, ^^^ p < 0.001, ^^^^ p < 0.0001 vs. corresponding 8Gy + miR-ve inhibitor group. miR-711 inhibition attenuates irradiation-induced downregulation of DNA repair molecules Rad50 and Rad54l2.

Figure 8

miR-711 inhibition attenuates irradiation-induced downregulation of DNA repair molecules Rad50 and Rad54l2. qPCR quantification of RAD50 and RAD54l2 (A). Rad50 mRNA (F(10,22) = 43.49, p < 0.0001 at 30 min and 1 h, p = 0.0082 at 3 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0039 at 3 h, p < 0.0001 at 6 h). Rad54l2 mRNA (F(10,22) = 39.1, p = 0.0005 at 30 min, p = 0.006 at 1 h, p = 0.0396 at 3 h after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p < 0.0001 at 3 h, p = 0.0007 at 6 h). n = 3/group for all groups, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. control; ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001, ^^^^ p < 0.0001 vs. corresponding 8Gy + miR-ve inhibitor group. RCNs were exposed to 8Gy and, after 3 h, cells were collected and lysed. One part of the lysate from each sample was used for RNA isolation and qPCR analysis for levels of miR-711, RAD50 and RAD54l2 (B). miR-711 (T(4) = 3.856, p = 0.0091), Rad50 mRNA (T(4) = 7.944, p = 0.0007), Rad54l2 mRNA (T(4) = 0.74, p = 0.0002). The other part was subjected to RIP using Ago2 antibodies, followed by qPCR analysis for levels of miR-711, RAD50 and RAD54l2 in the RISC (C). miR-711 (T(4) = 9.77, p = 0.0003); Rad50 mRNA (T(4) = 9.55, p < 0.0001); Rad54l2 mRNA (T(4) = 16.8, p < 0.0001). n = 3/group. Significance assigned by one-tailed t-test, *** p < 0.001, **** p < 0.0001 vs. control RCNs. This experiment was repeated with miR-711 inhibitor and miR-ive inhibitor. qPCR analysis for levels of miR-711, Rad50 and Rad54l2 (D): miR-711 (F(2,6) = 41.13, p = 0.0008 after IR with miR-ve inhibitor, compared to control), Rad50 mRNA (F(2,6) = 79.83, p < 0.0001 after IR with miR-ve inhibitor, compared to control; for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0200), Rad54l2 mRNA (F(2,6) = 41.51, p = 0.0003 after IR with miR-ve inhibitor, compared to non-irradiated control; for miR-711 inhibitor, compared to miR-ve inhibitor, p = 0.0016). n = 3/group, *** p < 0.001, **** p < 0.0001 vs. control; ^ p < 0.05, ^^ p < 0.01 vs. corresponding 8Gy + miR-ve inhibitor group.

Figure 9

Representative microscopy images from 30 min, 6 and 24 h of RCNs stained for γ-H2A.X (red), and DAPI (blue) (A). Representative microscopy images from 30 min, 6 h and 24 h of RCNs stained for 53BP1 (red), and DAPI (blue) (B). Data were calculated and plotted for all fields together as a cumulative frequency distribution without binning for γ-H2A.X foci count (C) and nuclear staining intensity (D). Data were calculated and plotted for all fields together as a cumulative frequency distribution for 53BP1 foci count (E). For all three parameters (L,M,n), samples treated with 8Gy + miR-ve inhibitors were significantly different from controls at all time points ( p < 0.0001, G:H(7)= 2257, H:H(7) = 2588, I:H(7) = 1041). Irradiated samples transfected with miR-711 inhibitor were significantly different from those transfected with miR-ve inhibitors at 6 h and 24 h for all parameters ( p < 0.0001) but not at 30 min.

Figure 10

The schematic illustration of the role of miR-711 and miR-23a-3p on the IR-induced neuronal outcome. Pro-apoptotic events are shown in red, anti-apoptotic events are shown in green.