Regulatory roles of miR-22/Redd1-mediated mitochondrial ROS and cellular autophagy in ionizing radiation-induced BMSC injury

Abstract

Ionizing radiation (IR) response has been extensively investigated in BMSCs with an increasing consensus that this type of cells showed relative radiosensitivity in vitro analysis. However, the underlying mechanism of IR-induced injury of BMSCs has not been elucidated. In current study, the regulatory role of miR-22/Redd1 pathway-mediated mitochondrial reactive oxygen species (ROS) and cellular autophagy in IR-induced apoptosis of BMSCs was determined. IR facilitated the generation and accumulation of mitochondrial ROS, which promoted IR-induced apoptosis in BMSCs; meanwhile, cellular autophagy activated by IR hold a prohibitive role on the apoptosis program. The expression of miR-22 significantly increased in BMSCs after IR exposure within 24 h. Overexpression of miR-22 evidently accelerated IR-induced accumulation of mitochondrial ROS, whereas attenuated IR stimulated cellular autophagy, thus advancing cellular apoptosis. Furthermore, we verified Redd1 as a novel target for miR-22 in rat genome. Redd1 overexpression attenuated the regulatory role of miR-22 on mitochondrial ROS generation and alleviated the inhibitive role of miR-22 on cell autophagy activated by IR, thus protecting BMSCs from miR-22-mediated cell injury induced by IR exposure. These results confirmed the role of miR-22/Redd1 pathway in the regulation of IR-induced mitochondrial ROS and cellular autophagy, and subsequent cellular apoptosis.

Fig. 1. Induction role of ionizing radiation on ROS generation.

a Intracellular total ROS staining and antioxygenation validation of NAC in BMSCs at 24 h post-IR. b Fluorescence intensity detection of intracellular ROS. c Mitochondrial ROS staining and antioxygenation validation of MitoQ in BMSCs at 24 h post-IR. d Ratio analysis of MitoSox Red/MtioTracker Green and validation of antioxygenation of MitoQ in BMSCs at 24 h post-IR. (**p ≤ 0.01; ***p ≤ 0.001)

Fig. 2. Activation role of IR on cellular autophagy and its regulatory effect on intracellular and mitochondrial ROS generation.

a Analysis of Ad-mCherry-LC3B. b Protein expression of autophagy-related markers (LC3, Atg7) and interventive effect validation of autophagic agonist and inhibitor at 24 h post-IR. c, d Fluorescence intensity detection of intracellular ROS and mitochondrial ROS staining after Rapamycin and 3-MA intervention. e Ratio analysis of MitoSox Red/MitoTracker Green following autophagy intervention. (* ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

Fig. 3. The regulatory role of radiation-induced mitochondrial ROS and autophagy on cellular apoptosis.

a JC-1 analysis of mitochondrial membrane potetial at 24 h post-IR. b Caspase-3 activity detection following MitoQ intervetion and sebsequent IR exposure. c Caspase-3 activity detection following autophagic intervetion and sebsequent IR exposure. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

Fig. 4. Radiation-induced expression of miR-22.

a miR-22 expression at 0、2、4、8、12、24 h after 6 Gy radiation. b PCR verification of transfection efficiency of miR-22 mimics and inhibitor. (***p ≤ 0.001)

Fig. 5. Regulatory role of miR-22 on the radiation-induced generation of ROS.

a Intracellular total ROS staining in BMSCs following miR-22 modification and sebsequent IR. b Fluorescence intensity detection using flow cytometry. c Mitochondrial ROS staining in BMSCs following miR-22 modification and sebsequent IR. d Ratio analysis of MitoSox Red/MitoTracker Green. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

Fig. 6. Regulatory role of miR-22 on the radiation-induced damage to intracellular antioxidant system and autophagy activation.

a The ratio of GSH to GSSG in rBMSCs treated with miR-22 modification and IR exposure. b Mitochondrial SOD detection. c Protein expression of autophagy-related markers at 24 h after miR-22 modification and radiation. d Ad-mCherry-LC3B analysis. e–g Relative expression of autophagic related protein (Atg7, LC3, Atg12) under miR-22 modification and subsequent 6 Gy IR exposure. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

Fig. 7. Regulatory role of miR-22 on the radiation-induced cellular apoptosis.

a JC-1 analysis of mitochondrial membrane potetial at 24 h after miR-22 modification and radiation. b Mitochondrial-mediated apoptosis-related protein (Bcl-xl, Bak, Caspase-9, Cyto C, Bax) expression. c Caspase-3 activity analysis of rBMSCs following miR-22 transfection and subsequent IR. (**p ≤ 0.01; ***p ≤ 0.001)

Fig. 8. Dual luciferase report assay of miR-22 with Redd1.

a Prediction of the binding site between miR-22 and Redd1. b miRNA vector construction. c The influnce of miR-22 on the protein expression of Redd1. d Quantitative analysis of protein expression. e, f Luciferase activity detection at 24 and 48 h following miR-22 and Redd1 transfection. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

Fig. 9. Verification of Redd1 genetic modification.

a PCR analysis of transfection efficiency of siRNA. b Protein analysis of transfection efficiency of siRNA at 48 h post-transfection. c Relative protein expression of Redd1 following siRNA intervention. d PCR analysis of transfection efficiency of Redd1 overexpression. e Protein analysis of transfection efficiency of Redd1 overexpression at 48 h post-transfection. f Relative protein expression of Redd1 following overexpression intervention. (*p ≤ 0.05; **^/##p ≤ 0.01; ***^/###p ≤ 0.001)

Fig. 10. Regulatory role of miR-22/Redd1 on radiation-induced generation of mitochondrial ROS and damage to intracellular antioxidant system.

a Mitochondrial ROS staining. b Ratio analysis of MitoSox Red/MitoTracker Green. c, d GSH/GSSG and mitochondrial SOD analysis in rBMSCs following miR-22, Redd1 modification and subsequent IR exposure. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

Fig. 11. Regulatory role of miR-22/Redd1 on radiation-induced cellular autophagy.

a Protein expression of autophagy-related markers at 24 h after Redd1/miR-22 modification and radiation. b Ad-mCherry-LC3B analysis. c (T.E.M analysis) Black arrow represents the autophagosome

Fig. 12. The negative regulation of Redd1 on mTORC1 activity.

a Protein expression of mTORC1 downstream targets p70 S6 Kinase (p70-S6K). b Relative protein expression of p-p70-S6K. (**p ≤ 0.01; ***p ≤ 0.001)

Fig. 13. Regulatory role of miR-22/Redd1 on radiation-induced cellular apoptosis.

a JC-1 analysis of mitochondrial membrane potetial at 24 h after genetic modification and subsequent radiation. b Mitochondrial-mediated apoptosis-related protein expression. c Caspase-3 activity analysis. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001)

Fig. 14

Molecular mechanism diagram