SUMO1 Modification Of C/EBP? And Its Effect On Proliferation And Differentiation In Human AEC?

ObjectiveThe CCAAT enhancer binding protein alpha(C/EBP?)is essential for lung development and promotes lung differentiation and maturation.The C/EBP? mainly expressed in AEC? in the lung.Small ubiquitin-like modifier(SUMO),as an important post-translational modification of proteins,can regulate the transcription factor activity,protein subcellular localization,and DNA damage repair.In mammals,SUMO1,SUMO2/3,and SUMO4 four SUMO subtypes exist.Among them,SUMO1 mainly exists in combination with other compounds,and SUMO2/3mainly exists in free form.This study first identified whether C/EBP? could be modified by SUMO1 in AEC? and identified its modification site,and further studied the effect of SUMO1 modified C/EBPa on the proliferation and differentiation of AEC?.Method(1)To detect the colocalization expression of C/EBP? and SUMO1 in human AEC?,immunofluorescence double labeling was used.(2)Co-IP was used to detect the interaction of C/EBP? and SUMO1 in AEC?.(3)SUMOsp 2.0 software predicted that the action site of human C/EBPa modified by SUMO1 is Lysine 161(K161).AEC? was transfected by wild-type GFP-C/EBP? plasmid and the mutant GFP-C/EBP?-K16 IR plasmid,then CO-IP was used to identify the SUMOylation site.(4)AEC? was transfected by wild-type GFP-C/EBP? plasmid,mutant GFP-C/EBP?-K16 IR plasmid and PCDNA3.0,then total protein was extracted,the expression of C/EBP? was detected by Western blot and AQP5 was detected by flow cytometry,CCK-8 method was used to detect the cell colonization.Result(1)Immunofluorescence double labeling revealed that there is colocalization between SUMO1 and C/EBP? in AEC?,and it was mainly localized in the nucleus.C/EBP? and SUMO1 can show the C/EBP?-SUMO1 interaction strip Band,suggesting that C/EBP? can interact with SUMO1.In wild-type GFP-C/EBP? transfected AEC?,GFP-C/EBP?-SUMO1 specific band can be detected;and mutant GFP-C/EBP? transfection In the AEC?,the GFP-C/EBP?-SUMO specific band could not be detected,suggesting that the SUMOylation modification site ofC/EBP? is the 161 th lysine of C/EBP?.(2)Compared with the control group,the percentage of AQP5+ was increased in the transfected wild-type C/EBP? group and the transfected mutant C/EBP?-K161 R group,and compared with the transfected wild-type C/EBP ? group.The percentage of AQP5+ in the C/EBP ?-K161 R mutant group was significantly lower(P<0.05).Compared with the control group,the cell proliferation was transfected into wild-type C/EBP ? group and transfected mutant C/EBP?-K161 R group.The index CCK-8 was decreased,and the cell proliferation index CCK-8 was significantly increased after AEC? transfection of C/EBP?-K161 R mutant plasmid compared with transfected wild-type C/EBP? group(P<0.05).).Conclusion(1)C/EBP? in human AEC? can be modified by SUMO1 and its modification site is lysine at position 161 of C/EBP?.(2)SUMO1 modification may play a negative regulatory role in C/EBP?-mediated AEC? proliferation and differentiation.