Mechanistic Study On Peroxidase Prdx6 In Regulation Of Skin Response After Ionizing Radiation

Objective Radiation-induced skin injury is a common complication after tumor radiation therapy,nuclear accident and bone marrow transplantation.However,the molecular mechanism underlying the pathogenesis of radiation-induced skin injury has not been extensively reported and it lacks effective prevention and treatment.The objectives of this project are I.High-throughput screening of key molecules associated with radiation-induced skin injury.II.Mechanism of miR-214/Prdx6 involving in the regulation of radiation-induced skin injury.III Prdx6 as a potential therapeutic target of radiation-induced skin injury verified by animal model.Methods I.High-throughput screening of key molecules associated with radiation-induced skin injury.1.Two-dimensional gel electrophoresis combined with mass spectrometry was used to screening differentially expressed protein induced by ionizing radiation.2.miRNA microarray was used to screening differentially expressed miRNA induced by ionizing radiation.3 Screening the potential mechanism of radiation-induced skin injury by analyzing the protein and miRNA profiles using bioinformatics tools.II.Mechanism of miR-214/Prdx6 involving in the regulation of radiation-induced skin injury.HaCaT cells were transfected with the Prdx6 expression vector.1.Luciferase assay and western-blot assay were conducted to identify whether Prdx6 was a target gene of miR-214.2.clonogenic assay was used to test the radiosensitivity of HaCaT cells.3.DCF-DA assay was used to detect ROS level of HaCaT cells and Mito-tracker Red were used to examin the damage of mitochondria.4.Flow Cytometry was used to detect cell apoptosis rate.5.Luciferase assay was used to verify the transcriptional level response mechanism of Prdx6.III Prdx6 as a potential therapeutic target of radiation-induced skin injury verified by animal model.SD rats were transfected with the Prdx6 expression vector.1.Animal model of radiation induced skin injury of SD rats were constructed.2.DCF-DA assay was used to detect tissue ROS level and thiobarbituric acid(TBA)assay was used to detect tissue MDA level.3.H&E staining was used to detect skin histopathology.Results We found 24 preferentially expressed proteins and 12 dysregulated miRNAs in irradiated skin.By analyzing the protein and miRNA profiles using bioinformatics tools,we identified a possible interaction between miR-214 and peroxiredoxin-6(Prdx6)Next,we investigated the expression of Prdx6 and the consequences of its dysregulation.Prdx6 is suppressed by radiation-inducible miR-214 and is involved in the pathogenesis of radiation-induced skin injury.Overexpression of Prdx6 conferred cells radioresistance,decreased cell apoptosis and preserved mitochondrial integrity after radiation exposure and the results were statistically significant.In addition,in vivo transfection with Prdx6 reduced radiation-induced reactive oxygen species(ROS)and the malondialdehyde(MDA)concentration and ameliorated radiation-induced skin damage in rats.Our present findings illustrate the molecular changes during radiation-induced skin injury and the important role of Prdx6 in ameliorating this damage in rats.Conclusion In this thesis,Prdx6 and miR-214 were found to be key molecules in skin tissue induced by ionzing radiation and confirmed that Prdx6 is a novel target gene of miR-214.Inhibition of miR-214 or overexpression of Prdx6 can reduce the sensitivity of skin cells/tissue to radiation damage and reduce ROS levels and MDA concentrations in cells/tissues,indicating that the miR-214/Prdx6 pathway plays an important role in radiation-induced skin injury.This paper suggested that Prdx6 as a potential target for the prevention of radiation skin injury which provided new insights and methods for the prevention and treatment of the disease.