Dexamethasone Upregulate The Expression Of Aquaporin4 In A549 Cells By Promoting SUMOylation

Purpose:1.To clarify the effect of dexamethasone on the expression of AQPs.2.To explore the effect of dexamethasone on SUMOs expression.3.To explore the regulatory relationship between sumo modification and AQPs.Methods:1.Effects of dexamethasone on the growth of A549 cells and on the expression of AQPs.A549 cells were selected as the research object to replace human alveolar type II epithelial cells.After adding different concentrations of dexamethasone cultivated for different time,the images of the cells were observed under microscope,and the vitality and apoptosis of A549 cells were detected by CCK-8 and TUNNEL assay.The expression levels of GR,AQP1,AQP4 and AQP5 proteins in A549 cells were detected by Western Blot.2.Effects of dexamethasone on the expression of SUMO1,SUMO2/3 and UBC9.After cultivating with different concentrations of dexamethasone for different time,the expression levels of SUMO1,SUMO2/3 and UBC9 proteins in A549 cells were detected by Western Blot.3.After cultivating with dexamethasone,the temporal and spatial distribution of SUMOs and AQPs were detected,and the expression of AQPs was detected after knockdown of SUMO2/3 in A549 cells.After cultivating with dexamethasone for different time,the temporal and spatial distribution of SUMO1,SUMO2/3,GR,AQP1,AQP4 and AQP5 were detected by immunofluorescence staining in A549 cells.SUMO2/3 interference plasmid was constructed and transfected into A549 cells.The interference effects of SUMO2 and SUMO3 were detected by PCR and the expression levels of SUMO2/3,UBC9,AQP1,AQP4 and AQP5 were detected by Western Blot.Results:1.Microscopically,it was found that after the treatment of high concentration of dexamethasone,the morphology of A549 cells did not change significantly,the arrangement was sparse and the number decreased,but the change of A549 cells was not obvious after the treatment of low concentration of dexamethasone.The results of CCK-8 and TUNNEL showed that low concentration of dexamethasone had no significant inhibitory effect on the growth of A549 cells.After cultivating with dexamethasone,the expression of GR gradually increased and reached the peak at 36 hours.The expression of AQP1 had no significant change.The expression of AQP4 increased gradually and reached the peak at 48 hours.The expression of AQP5 increased gradually,reached the peak at 24 hours,and then decreased to the normal level.2.The results of Western Blot showed that after treatment with dexamethasone,the SUMO1 in the binding state did not change significantly,while the SUMO1 in the free state increased slightly,and the SUMO2/3 in the binding state increased significantly.It was statistically significant at the concentrations of 10^-7,5x10^-77 and10^-8M.There was no significant change in free SUOMO2/3.So 10^-77 M was selected as the concentration of the follow-up experiments.The expression levels of SUMO1,SUMO2/3 and UBC9 proteins were detected again.It showed that dexamethasone had no significant effect on SUMO1 and UBC9,while the SUMO2/3 increased.3.The results of immunofluorescence staining showed that after treatment with dexamethasone,plasma nucleus translocation occurred in SUMO1 and SUMO2/3,the expression of AQP1 and AQP4 increased,and the expression of AQP5 increased in24 hours and decreased in 48 hours.GR followed the same law as AQP5.PCR results showed that the expression of SUMO2 and SUMO3 decreased after SUMO2/3 interference plasmid was transfected into A549 cells.The results of Western Blot showed that after knockdown of SUMO2/3,the expression of AQP4decreased while SUMO2/3 decreased.Conclusion:Dexamethasone did not affect the growth of A549 cells,but could up-regulate the expression of AQP4 and activate SUMO2/3.After knockdown of SUMO2/3,the expression of AQP4 decreased.