| Objective: Co ? irradiates pulmonary epithelial cells and then perform mi RNA sequencing,screen mi RNAs with significant differential expression and explore their biological functions that may be involved in the development of pulmonary fibrosis.Select a mi RNA and explore its role and mechanism in radiation-induced pulmonary fibrosis through in vivo and in vitro experiments.Methods: 6 Gy 60 Co ? irradiated A549 cells and BEAS-2B cells,observed the cell morphology with an inverted microscope at 0 h,6 h,and 48 h after irradiation,and extracted cell proteins at these three time points,the contents of EMT-related proteins such as E-cadherin,N-cadherin,Vimentin,?-SMA and Twist were verified by Western blot.6 Gy 60 Co ? irradiated BEAS-2B cells,and the cells were continuously cultured,then the cellular RNA was extracted at 0 h,6 h,and 48 h after irradiation,and mi RNA sequencing was performed.The sequencing results were analyzed.The differentially expressed mi RNAs were screened by Real-Time PCR.At the same time,by analyzing KEGG and GO,we explored the biological functions and pathways of these differentially expressed mi RNAs that may be involved in the occurrence and development of EMT and RIPF.Through cell transfection experiments,transfection of mi RNA mimics or mi RNA inhibitors can overexpress or knock down mi RNAs in cells,than the changes of EMT-related proteins in transfected cells were verified by Western blot and immunofluorescence.We predicted mi RNA downstream target genes through Target Scan,mi RTar Base and Gene Cards,verificated of changes in EMT-related proteins and target gene content after irradiation and transfection of mi RNA mimic,mi RNA inhibitor and target gene interference sequence(si RNA)by Western blot,Real-Time PCR and immunofluorescence.Double luciferase reporter experiment confirms direct interaction between mi RNA and target gene.Western blot verified the protein content of EMT-related signaling pathways that mi RNA target genes may participate in,and further clarified the mechanism of mi RNAs involved in radiation-induced EMT.Adeno-associated virus carrying mi RNA was injected into the pulmonary tissue of C57 mice by nasal administration,and then the lungs of the mice were locally irradiated with 25 Gy 60 Co ? and the mouse pulmonary tissue collected at 1month,2 month,3month and 4 month after irradiation.We extracted RNA and protein of pulmonary tissue,and verifyed pulmonary fibrosis-related proteins collagen and ?-SMA content by Western blot.At the same time,pathological sections of mouse lung tissue were prepared,and HE and Masson staining were performed.HE staining was quantified by the pulmonary fibrosis score sheet.Masson staining was analyzed by Image Pro Plus software to further explore the role and mechanism of mi RNA in RIPF.Results: 6 Gy 60 Co ? irradiated A549 cells and BEAS-2B cells and found that the cell morphology changed from epithelial cell morphology to mesenchymal cell morphology.Western blot verification of EMT-related proteins showed that E-cadherin,a protein marker of epithelial cells,was reduced.Interstitial cell markers N-cadherin,Vimentin,?-SMA and Twist were significantly increased.Analysis of the sequencing results revealed that irradiation can up-or down-regulate the expression of many mi RNAs,The verification results of hsa-mi R-127-3p,hsa-mi R-129-5p,hsa-mi R-150-5p hsa-mi R-155-5p,hsa-mi R-451 a,hsa-mi R-342-3p,hsa-mi R-3195,hsa-mi R-9-5p,hsa-mi R-381-3p,hsa-mi R-582-3p,hsa-mi R-32-5p,hsa-mi R-199a-5p are consistent with the sequencing results.Functional analysis of KEGG and GO revealed that many m RNAs of differentially expressed mi RNAs were enriched in the signaling pathways involved in the development of EMT,such as the Wnt signaling pathway and the MAPK signaling pathway.Through further analysis of sequencing results and verification results,and through a large number of literature surveys,we selected mi R-155-5p as the research object.After irradiation,mi R-155-5p expression was down-regulated.After knocking down mi R-155-5p in A549 and BEAS-2B cells,the cells developed EMT.When mi R-155-5p was overexpressed in cells,it can reverse the EMT phenomenon produced by IR.GSK-3? is a possible target gene of mi R-155-5p.The two are negatively correlated,and GSK-3? is significantly increased in the cells after irradiation.The double luciferase reporter gene experiment also confirmed that GSK-3? is mi R-155-5p direct target gene.When we knocked down GSK-3? in cells,we can reverse the EMT phenomenon that occurs after IR or knockdown mi R-155-5p.NF-?B did not change significantly when mi R-155-5p or IR was knocked down,but phosphorylated NF-?B increased significantly.When we overexpress mi R-155-5p or knock down GSK-3?,we can reverse the increased phosphorylated NF-?B.In vivo experiments,HE and Masson staining confirmed that with the increase of time after irradiation,the degree of fibrosis in the pulmonary tissue of the irradiation group of mice gradually increased,pulmonary tissue of mice injected with adeno-associated virus carrying mi R-155-5p before irradiation was less fibrotic.The same results were obtained at the protein level,that is,the collagen and ?-SMA in the pulmonary tissue of the mice in the irradiation group were significantly increased,while in the mice overexpressing mi R-155-5p,the levels of collagen and ?-SMA were reduced.Conclusion:1.6 Gy 60 Co ?-rays can induce EMT in A549 cells and BEAS-2B.2.Irradiation can induce hsa-mi R-127-3p,hsa-mi R-129-5p,hsa-mi R-150-5p hsa-mi R-155-5p,hsa-mi R-451 a down-regulation,And these mi RNAs have the same change trend at 6 h and 48 h,hsa-mi R-9-5p,hsa-mi R-381-3p were down-regulated at 6 h,hsa-mi R-342-3p,hsa-mi R-199a-5p and hsa-mi R-32-5p were down-regulated at 48 h,hsa-mi R-381-3p and hsa-mi R-582-3p were up-regulated at 48 h.3.Iradiation can reduce mi R-155-5p content in cells,and participate in the regulation of EMT through GSK-3? / NF-?B signaling pathway.4.Overexpression of mi R-155-5p can inhibit the activation of GSK-3? / NF-?B signaling pathway,thereby inhibiting the occurrence of RIPF.5.25 Gy irradiation can cause RIPF in C57 mice,reduce mi R-155-5p content in the lung tissue of mice,and participate in the regulation of the occurrence and development of RIPF. |
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