The Effects Of MiRNA Let-7i On Radiation-induced Pulmonary Epithelial-mesenchymal Transition

Objective Radiation-induced pulmonary fibrosis(RPF)is the most serious one,which threatens the life of the patient health.Currently,mechanism of RPF is unclear.Studies have shown that ionizing radiation(IR)can induce epithelial-mesenchymal transition(EMT)of lung epithelial cells and then transformed into fibroblasts,eventually inducing RPF.The presently available evidence suggests that mi RNAs targeting EMT account for their effects on pulmonary fibrosis.Therefore,this study verifies whether IR can induce EMT at first,and further explores whether IR can regulate EMT through some mi RNAs.We hope to screen out a mi RNA that plays an important role in ionizing radiation-induced EMT and elucidate its mechanism to provide new ideas for research of RPF.Methods A549 cells and Beas-2B cells were irradiated with 60Coγ ray 6Gy,C57BL/6 mice were irradiated with 60Coγ ray 20 Gy to extract alveolar epithelial cells type II(AECII)from the lung tissue,Western blot(WB)and flow cytometry were used to detect the changes of EMT related markers in the three types of cells.Mi RNA microarrays were used to detect the differentially expressed mi RNAs in the lung tissues of the C57 mice at 14 days after irradiation,and let-7i,which had undergone significant changes in the lungs,was selected as the study object.The expression of let-7i in irradiated A549 cells and the transfection efficiency of let-7i mimics and inhibitors were detected by RT-q PCR.WB was used to detect the expression of EMT-related proteins after transfection.The target protein of let-7i was predicted by the mi RNA target gene prediction website,and IL-10 was selected for validation.RT-q PCR,WB and dual luciferase reporter assays were used to detect the target interaction between let-7i and IL-10.The expression of EMT related markers and EMT pathway proteins AKT and p-AKT were detected by WB after IL-10 si RNA was transfected into A549 cells and Beas-2B cells,as well as in irradiated A549 cells after knockdown of let-7i and IL-10.Results The expression of E-cadherin were decreased and N-cadherin were increased in A549 cells and Beas-2B cells after irradiation.The proportion of E-cadherin+ N-cadherin+ and ZO-1+ N-cadherin+ cells in A549 cells,Beas-2B cells and AECII was significantly increased.The expression of let-7i in lung tissue of C57 mice and A549 cells were significantly increased after irradiation.Overexpression of let-7i promoted the expression of E-cadherin and prevented the expression of N-cadherin,Vimentin,twist and p-AKT,but the expression of AKT was not changed.Let-7i suppression prevented radiation-induced increases in E-cadherin level and decreases in N-cadherin,Vimentin,twist and p-AKT levels in A549 cells.The expression of IL-10 m RNA and protein were decreased after overexpression of let-7i,and were increased after knocking down let-7i.Dual luciferase reporter assays showed that after overexpression of let-7i,luciferase activity was significantly reduced in the IL-10 wild-type group,whereas no change was observed in the mutant group.The knockdown of IL-10 reduced the expression of E-cadherin and increased the expression of N-cadherin,Vimentin,twist and p-AKT in A549 cells and Beas-2B cells.The expression of E-cadherin in irradiated A549 cells with knockdown of let-7i and IL-10 were less than in knockdown of let-7i alone,the expression of N-cadherin,Vimentin,twist,and p-AKT were more than in knockdown of let-7i alone.There was no significant change in the expression of AKT in each groupConclusion IR can induce EMT in lung epithelial cells and induce let-7i overexpression at animal and cell level.Let-7i can regulate radiation-induced EMT by inhibiting the expression of target protein IL-10 and activating PI3 K / Akt pathway;let-7i / IL-10 / PI3 K / Akt is a new way of radiation-induced EMT.