The Role Of AFAP1 In Senescent A549 Cells Induced By Bleomycin

Objective Senescence is an irreversible and persistent inhibition of cell proliferation under stress.Different from apoptosis,senescent cells can resist apoptosis and survive for a long time.When senescent cells cannot be effectively cleared by immune cells,they accumulate in tissues over time,and secrete too many cytokines to change the niche in the tissue.This effect will affect the differentiation and growth of the surrounding cells and lead to the deterioration of tissues and organs,which will promote aging and aging-related diseases.Idiopathic pulmonary fibrosis is a disease associated with the senescence of alveolar type 2 epithelial cells.However,the molecular mechanisms involving how senescence of alveolar type 2 epithelial cells contribute to idiopathic pulmonary fibrosis remain unclear.The results of gene screening revealed that AFAP1 was down-regulated in the lungs of mice with radiation-induced lung fibrosis.However,A549 cells can be induced senescence by bleomycin.Therefore,the molecular mechanism of senescent alveolar type 2 epithelial cells was explored in the bleomycin-induced A549 cell senescence model.Murine model of pulmonary fibrosis was established by bleomycin in vivo to research the mechanism of AFAP1 in senescent lung cells.Methods A549 cells were treated with 50 ug/ml bleomycin for 5 days to establish a cellular senescence model.The cells were treated with the same concentration of bleomycin for 1-5 days to observe the whole process from cell cycle arresting to senescence,and SA-β-Gal staining was used to detect the number of senescent cells and count the area of senescent cells.Western blot and immunofluorescence assay were used to detect the expression of AFAP1,p21,c-Src and other proteins.After overexpression of AFAP1,the changes of cell senescence and expression of each protein were observed.Binding statement of AFAP1 and c-Src under different treatments was observed by immunoprecipitation.The expression of several cytokines at the protein level and mRNA level was detected by ELISA and qRT-PCR,respectively,and the viability of senescent cells was detected by MTT assay.The C57BL/6J mice were divided into control group and BLM group,and the bleomycin treated group was administered at a dose of 0.04 mg/100 u1/2 times per week for 8 times.Then,the lung index was measured and collagen deposition in lung tissue,hydroxyproline content in lung,pathological score of lung tissue were determined.Likewise,co-immunoprecipitation was used to observe the binding status of AFAP1 and c-Src under different treatments.Results After treatments with 50 ug/ml bleomycin for 5 days,a cell senescence model was established.The expression of SA-β-Gal positive cells in the BLM group increased and the cell volume significantly expanded;the expression level of p21 increased too;the expression level of AFAP1 declined.This was also consistent with the results of immunofluorescence.The expression levels of AFAPI and activated c-Src(Src pY416)were consistent in senescent A549 cells.The expression of the BLM group markedly decreased on the 4th day and went the lowest on the 5th day.The expression level of c-Src and Src pY527)was unchanged.After overexpression of AFAP1 and induction with bleomycin,there was no significant difference in the number of SA-P-Gal positive cells and the volume of cells compared with the control group.There was no difference between Src pY416 and p21.However,overexpression of AFAP1 reduced the secretion of TGFβ,TNFa,and IL-6,and enhanced the viability of bleomycin-induced senescence of A549 cells.After we had transfected A549 cells with AFAP1 plasmids,the two proteins did not interact together under normal conditions,but the binding capacity of the two proteins increased during senescence.In this murine model of bleomycin induced pulmonary fibrosis,AFAP1 expression was downregulated and the ability of binding with Src pY416 was enhanced comparing to the control group.Conclusions The expression of AFAP1 was down-regulated and the c-Src activity decreased in senescent A549 cells.Overexpression of AFAP1 could not protect the A549 from senescence,but it could increase the viability of senescent cells and reduce the secretion of cytokines in senescent cells.This would alsobe likely to reduce the cellular senescence-associated phenotype by affecting the interaction of AFAP 1 and c-Src.However,the roles and mechanisms between AFAP1 and c-Src interactions in the development of cellular senescence need further study.