Interaction Between Sumoylation And Phosphorylation Of P65

Objective:Liver cancer is one of the most common malignancies in the world,affecting the whole world.Hepatocellular carcinoma（HCC）is the main histological type of primary hepatocellular carcinoma.Post-translational modification（PTM）are modifications that occur in many key signaling pathways during tumor transformation.Phosphorylation is a widely studied post-translational modification of proteins in hepatocellular carcinoma.It has been reported that phosphorylation of p65 is associated with cancer and promotes the progression of cancer.The preliminary results of our research group found that SUMO1 modification of p65 promoted the progression of hepatocellular carcinoma.The purpose of this study was to investigate whether there is a relationship between the two post-translational modifications of p65 protein.If so,what kind of connection.Methods:Immunohistochemistry（IHC）is used to detect the expression of SUMO1 and phosphorylated p65 in human hepatocellular carcinoma tissues;subsequently,mutants of SUMO1-related p65 sumo-mediated sites are constructed and CO-IP（CO immunoprecipitation）Immunoprecipitation and Western Blot were used to detect the modification sites of the mutants at the SUMO site of p65.Luciferase was used to detect the relationship between the mutants at the SUMO site of p65 and NF-κB transcriptional activity.MTT and cell invasion assay were used to verify the influence on malignant biological behavior of hepatocellular carcinoma cells.Software was used to predict the phosphorylation mutation sites of p65（Ser276 and Ser536）,and then the corresponding mutant plasmid was constructed.After the successful construction of the mutant was confirmed by sequencing.The effects of phosphorylation of p65 on proliferation and invasion of hepatocellular carcinoma were evaluated by CCK8 cell proliferation and invasion assay.Immunoprecipitation was used to detect the effect of p65 phosphorylation on sumoylation of plasmids overexpressing wild-type plasmids and phosphorylation site mutations on hepatocellular carcinoma cell lines.p65wild-type plasmids and sumo-modified plasmids were overexpressed in hepatocellular carcinoma cell lines,and the effect of sumo-modified plasmids on their phosphorylation was detected by using antibodies to the phosphorylation site of p65.According to the model of sumo-modified regulation of transcription factor activity,inhibitory proteins can be recruited to inhibit the activity of transcription factors after sumo-modified transcription factors occur in the nucleus.SUMO1 mediated the entry of MANF into the nucleus,so the mutants of the sumo-mediated site of MANF were constructed.Cellular immunofluorescence was used to detect the effect of sumo-mediated site mutation on MANF localization.Luciferase was used to determine the effect of MANF mutation on NF-κB transcriptional activity.Results:1.The expression of SUMO1 in liver cancer is consistent with the phosphorylation of p65.Immunohistochemical results indicated that the expression of SUMO1 in HCC was consistent with the phosphorylation of p65 at S276,and the expression of SUMO1 was also consistent with the phosphorylation of p65 at S536.2.Construct mutants of p65 sumoylation site and verify the effect of the modification site and SUMO 1-related sumoylation of p65 on NF-κB transcriptional activity.Since nuclear transfer of p65 can activate NF-κB transcriptional activity,and SUMO1 protein can promote nuclear transfer of p65,we wanted to investigate whether SUMOylation of p65 affects NF-κB transcriptional activity.We constructed a plasmid mutated at the p65 sumo-modified site and found that NF-κB transcriptional activity was upregulated when the p65 sumo-modified site was mutated.These results indicate that SUMOylation of p65 inhibits NF-κB transcriptional activity,which is inconsistent with our previous hypothesis that SUMO1 promotes p65 nuclear transfer and upregulations of NF-κB transcriptional activity.We considered that in addition to SUMO1 protein,there is also SUMO 2/3protein in HCC cells.In order to investigate the SUMO1-related P65 suo modification,we increased the SUMO1-related sumo-modification level in HCC cells by overexpressing SUMO1 and knocking down SUMO2/3.In this case,the transcriptional activity of NF-κB was low after the mutation of the SUMO1-related p65 sumo-modification site,suggesting that the transcriptional activity of NF-κB was up-regulated by SUMO1-related p65 sumo-modification.3.The effect of SUMO1-related p65 sumoylation modification on malignant biological behavior of hepatoma cells.The transcriptional activity of NF-κB is related to the progression of hepatocellular carcinoma.Therefore,MTT cell viability assay and cell invasion assay were used to verify overexpression of SUMO1 or knockdown of SUMO2/3 to investigate the effect of SUMO1-related p65 suomodification on cells.The results showed that the cell viability and invasion ability were decreased after the SUMO1-related p65 suo modification site mutation.These results suggest that SUMO1-related modification of p65 promotes the progression of HCC.4.Construct plasmid of p65 phosphorylation site mutation and verify the effect of p65 phosphorylation on hepatocellular carcinoma.The constructed plasmid was transfected into Hep G2 hepatoma cell lines.Compared with the plasmid transfected with flag-p65-S276 A site mutation and flag-p65-S276,536 A common mutation,the proliferation ability of the cells was inhibited.However,the cell proliferation ability of flag-p65-S536 A transfected plasmid was not significantly changed compared with that of p65 wild-type plasmid.Cell invasion experiments showed that the invasion ability of cells transfected with mutant plasmid was inhibited to varying degrees.These results suggest that phosphorylation of p65 promotes the progression of liver cancer.5.Interactive regulation of sumoylation and phosphorylation of p65.The expression of p65 phosphorylation（site 276）was reduced after the mutation of the SUMO1-related sumoylation of p65,indicating that the SUMO1-related sumoylation of p65 promotes the phosphorylation of p65.After the phosphorylation site mutation of p65,the SUMO modification of p65 decreased,indicating that phosphorylation promotes its SUMO modification.6.Effects of sumoylation site mutation on MANF localization and NF-κB transcriptional activity.Inhibition of NF-κB transcriptional activity by MANF requires sumo-mediated MANF entry into the nucleus.After the mutation of THE sumoylation site of MANF,MANF was in cytoplasm.SUMO modification of MANF inhibits NF-κB transcriptional activity.Conclusion:The expression of SUMO1 is consistent with the phosphorylation expression of P65 in liver cancer tissues,and the SUMO1-related sumo1-related modification of p65 can promote liver cancer.Then,the construction of p65 phosphorylation site mutation plasmid and verification of p65 phosphorylation can also promote hepatocellular carcinoma.The interaction between sumoylation and phosphorylation of p65 showed that SUMO1-related sumoylation of p65 promoted the phosphorylation of p65 at Ser276,suggesting that the sumo1-related sumoylation of p65 promoted the phosphorylation of p65 at Ser276.After the phosphorylation site mutation of p65,SUMO1-associated p65 was reduced,and phosphorylation of p65 promoted SUMO1-associated p65 suo modification.Inhibition of NF-κB transcriptional activity by MANF only occurs after sumo-mediated MANF entry into the nucleus.