Study On The Mechanism Of Radiation-induced Lung Injury In Rats Based On Proteomics And Transcriptomics

Objective: This project aims to establish a rat model of radiation-induced lung injury(RILI)after treatment with different doses of radiation on right lung.In this study,we analyzed the differential expression proteins and genes related to RILI at protein and m RNA levels,explored the potential pathological mechanism of RILI,identified novel biomarkers and provided theoretical basis for the diagnosis of RILI.Methods: 1.45 SD rats were randomly divided into three groups for model construction.5rats without any treatment served as normal control.The other two groups containing 40 rats(20 rats for each group)were treated with 20 Gy or 30 Gy accumulated radiation dose targeting right lung,respectively.The right lung tissues of rats from each group were collected 1,3,7,14 and 28 days after radiation and pathological examination was performed.2.Proteomic analysis of RILI in rats: 3 rats 7-day after 30 Gy dose radiation treatment were used as the experimental group according to the pathology,and 3 rats without any treatment were used as the normal control.Proteins of right lung tissues from rats in two groups were extracted,through Protein Quality Test、TMT Labeling of Peptides、 Separation of fractions、LC-MS/MS Analysis、Proteomics Data analysis,Differentially expressed proteins were identified through GO analysis and KEGG pathway analysis.Analyze the function of differentially expressed proteins and the pathways involved.3.Transcriptomics analysis of RILI in rats: RNA of lung tissue from rats in the experimental and control group was extracted,RNA sequencing was performed using Illumina sequencing platform.Differential expressed genes were screened using DESeq2 software(1.20.0).Cluster analysis,GO and KEGG enrichment analysis were conducted to determine the signal transduction pathway and the biological process during the development of RILI.Finally,reverse transcription polymerase chain reaction(real time RT-PCR)was applied to verify the expression levels of key differential expressed genes.Results: 1.All the rats were alive after treatment with 20Gy/30 Gy radiation.The right lung of rats treated with radiation has developed different degree of pathological injury 3,7,14 and28 days after radiation compared with the normal control group.Pathological results showed that the RILI model of rat was successfully established.2.Proteomic analysis showed that 185 proteins were differentially expressed between the two groups,including 110 up-regulated proteins and 75 down-regulated proteins.The most upregulated protein is Tnc.Immunohistochemical results showed that Tnc was expressed in the extracellular matrix of lung tissue in the 30 Gy irradiation group.GO and KEGG enrichment analysis were performed to further clarify the functions of differentially expressed proteins,such as redox process and metabolic process.The signaling pathway involved in the the pathogenesis of RILI mainly included ECM receptor interaction,PPAR signal pathway and PI3K-Akt signal pathway.3.Transcriptomics analysis showed that there were 414 differentially expressed genes between the experimental group and the control group,including 270 up-regulated genes and 144down-regulated genes.GO function enrichment analysis showed that the differential genes were mainly involved in chromosome segregation and mitosis;The results of KEGG pathway analysis suggested that DEGs were involved in ECM receptor interaction,PI3K-Akt signaling pathways,c AMP signaling pathways and TNF signaling pathways.Key differentially expressed genes screened by KEGG pathway analysis including Fos,Cxcl2,Nr1d1,Angptl4,Ccn4,Tnc,which were further confirmed by real-time RT-PCR.The expression level of the above genes was subsequently tested by real-time RT-PCR,which was consistent with the trend of the results of transcriptome analysis.Conclusion: This study analyzed the function and signal transduction pathways of key proteins and genes of RILI in rats,and preliminarily revealed the potential mechanism of the development of RILI.We found that Fos,Cxcl2,Nr1d1,Angptl4,Ccn4,Tnc were closely related to RILI,which could be used as biomarkers and had the potential to be a novel tool for RILI diagnosis.In conclusion,our project provided an experimental basis for the treatment and research of RILI.