MicroRNAs Function In Ionizing Radiation-induced Epithelium-to-mesenchymal Transition(EMT) In Pulmonary Epithelial Cells

Objective:The aim of study is to identify expression profiles of miRNAs in the irradiated lung epithelial cells,and elucidate the role and mechanisms of the radiation-induced miRNA on the radiation-induced lung epithelial-mesenchymal transition.Methods:C57BL/6 mice were given a single dose of thoracic irradiation（20 Gy）with a ⁶⁰Coγ-rays source.The lung protein was extracted to observe the changes of EMT-related proteins,such as,E-cadherin,N-cadherin,Vimentin and Twist by Western blot analysis post14 days irradiation.The expression profiles of miRNAs in irradiated lung cells were identified by miRNA microarray analysis.A549 cells and BEAS-2B cells were irradiated with 6 Gyγrays.The morphology of the cells post 48 h radiation was observed with an inverted microscope,and the expression of EMT-related proteins was identified by Western blot.The target genes of differentially expressed miRNAs were predicted by TargetScan,miRDB and microT-CDS,combined with online tools such as DAVID,KEGG and DIANA.Functional acquisition/deletion experiments were performed in A549 and BEAS-2B cells by miRNA mimics and miRNA inhibitors to explore the role of miRNAs in IR-induced EMT.The candidate target genes that could play a roles in IR-induced EMT were confirmed by bioinformatics analysis,RT-qPCR,Western blot and dual luciferase reporter genes analysis.The mechanisms by which miRNAs mediate IR-induced EMT were further explored by detecting the activation of EMT-related pathway proteins.Results:When C57BL/6 mice were given a single dose of thoracic irradiation（20 Gy）with a ⁶⁰Coγ-rays source,EMT occurred in the lung tissues of mice post 14 day irradiation.Meanwhile,when A549 cells and BEAS-2B cells were irradiated with 6 Gyγrays for 48 h,the morphology of cells changed from epithelial to mesenchymal and the expression of E-cadherin decreased,and the expression of N-cadherin,Vimentin andα-SMA increased.The miRNA microarray analysis of mice lung tissue resulted in 10 differentially expressed mature mmu-miRNAs,of which 5were up-regulated and 5 were down-regulated.Species conservation analysis revealed 7 hsa-miRNAs,among which,hsa-miR-193a-3p,hsa-miR-150-5p,hsa-miR-362-3p and hsa-miR-21-5p was up-regulated,hsa-miR-541-5p,hsa-miR-486-3p and hsa-miR-34a-5p was down-regulated.Real-Time PCR confirmed that the expression changes of these 7 miRNAs in A549 and BEAS-2B after irradiation were consistent with the chip results.The KEGG pathway of miRNAs target gene is significantly enriched in EMT-related pathways including ErbB signaling pathway,TGF-βsignaling pathway,MAPK signaling pathway,Wnt signaling pathway and PI3K-Akt signaling pathway,suggesting that these 7 miRNAs may be potential regulatory molecules that regulate IR-induced EMT.MiRNA microarray analysis revealed that miR-21expression was up-regulated in irradiated lung tissue.We found that overexpression of miR-21 significantly promotes fibrotic EMT,and down-regulation of miR-21 prevents IR-induced EMT in lung epithelial cell fibrosis.PTEN was confirmed to be a direct target of miR-21 after irradiation.MiR-21 mimic significantly induced p-Akt,while anti-miR-21 significantly inhibited IR-mediated decreasing of PTEN and increasing of p-Akt.MiR-34a-5p was confirmed down-regulated post irradiation in A549 and BEAS-2B cells.Inhibition of miR-34a-5p greatly promoted EMT,while miR-34a-5p overexpression inhibited IR-induced EMT.It was confirmed that miR-34a-5p targeted regulation of CD44 and SNAI1 is up-regulated in irradiated lung epithelial cells and participates in IR-induced EMT.Moreover,inhibition of miR-541-5p significantly promoted EMT,while miR-541-5p overexpression inhibited IR-induced EMT.It was confirmed that miR-541-5p targeted regulation of SMAD2,SNAI2 and COL1A2 was up-regulated in irradiated lung epithelial cells and involved in IR-induced EMT.Conclusion:1.IR up-regulates the expression of miR-193a-3p,miR-150-5p,miR-362-3p and miR-21-5p,and down-regulates the expression of miR-541-5p,miR-486-3p and miR-34a-5p in lung epithelial cells.2.IR-induced miR-21 promotes IR-induced pulmonary fibrosis EMT via the miR-21/PTEN/Akt pathway.3.miR-34a-5p may inhibit IR-induced pulmonary EMT and RIPF by silencing CD44 and SNAI1.4.miR-541-5p may inhibit IR-induced pulmonary EMT and RIPF by silencing SMAD2,SNAI2 and COL1A2.