SUMO1 Modification Of C/EBPα And Its Effect On Proliferation And Differentiation In Human AECⅡ

ObjectiveThe CCAAT enhancer binding protein alpha(C/EBPα)is essential for lung development and promotes lung differentiation and maturation.The C/EBPα mainly expressed in AECⅡ 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/EBPα could be modified by SUMO1 in AECⅡ and identified its modification site,and further studied the effect of SUMO1 modified C/EBPa on the proliferation and differentiation of AECⅡ.Method(1)To detect the colocalization expression of C/EBPα and SUMO1 in human AECⅡ,immunofluorescence double labeling was used.(2)Co-IP was used to detect the interaction of C/EBPα and SUMO1 in AECⅡ.(3)SUMOsp 2.0 software predicted that the action site of human C/EBPa modified by SUMO1 is Lysine 161(K161).AECⅡ was transfected by wild-type GFP-C/EBPα plasmid and the mutant GFP-C/EBPα-K16 IR plasmid,then CO-IP was used to identify the SUMOylation site.(4)AECⅡ was transfected by wild-type GFP-C/EBPα plasmid,mutant GFP-C/EBPα-K16 IR plasmid and PCDNA3.0,then total protein was extracted,the expression of C/EBPα 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/EBPα in AECⅡ,and it was mainly localized in the nucleus.C/EBPα and SUMO1 can show the C/EBPα-SUMO1 interaction strip Band,suggesting that C/EBPα can interact with SUMO1.In wild-type GFP-C/EBPα transfected AECⅡ,GFP-C/EBPα-SUMO1 specific band can be detected;and mutant GFP-C/EBPα transfection In the AECⅡ,the GFP-C/EBPα-SUMO specific band could not be detected,suggesting that the SUMOylation modification site ofC/EBPα is the 161 th lysine of C/EBPα.(2)Compared with the control group,the percentage of AQP5+ was increased in the transfected wild-type C/EBPα group and the transfected mutant C/EBPα-K161 R group,and compared with the transfected wild-type C/EBP α group.The percentage of AQP5+ in the C/EBP α-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 α group and transfected mutant C/EBPα-K161 R group.The index CCK-8 was decreased,and the cell proliferation index CCK-8 was significantly increased after AECⅡ transfection of C/EBPα-K161 R mutant plasmid compared with transfected wild-type C/EBPα group(P<0.05).).Conclusion(1)C/EBPα in human AECⅡ can be modified by SUMO1 and its modification site is lysine at position 161 of C/EBPα.(2)SUMO1 modification may play a negative regulatory role in C/EBPα-mediated AECⅡ proliferation and differentiation.