| The small ubiquitin-related modification 1(SUMO1)is involved in the SUMOylation modification,which is an important post-translational modification of the proteins,and one of the most important functions of the SUMO1 protein is to mediate the translocation of the target proteins between nuclear and cytoplasm.P65 protein is one of the most important subunits of NF-?B.Phosphorylation of p65 activates NF-?B signaling pathway to promote the progression of hepatocellular carcinoma(HCC).It has been reported that phosphorylated p65 protein is related to its nuclear transfer.Our previous study found that there is an interaction between p65 and SUMO1 protein,overexpression of SUMO1 can promote the nuclear translocation of p65 protein,and SUMO1 can promote the progression of HCC.In addition,our group's previous experiments found that mesencephalic astrocyte-derived neurotrophic factor(MANF)has a suppressiveon HCC and there is a phenomenon of nuclear translocation in hepatoma cells.At the same time,it was confirmed that MANF interacts with the DNA binding domain of p65 protein in 293 T cells,inhibits the binding of p65 to the promoter of its target gene,thereby exerting the effect of inhibiting the NF-?B signaling pathway.Yeast two-hybrid experiments found that MANF can bind to SUMO protein,and the software predicts that MANF is the target protein of SUMO.However,whether there is a link between SUMO1 and p65 protein phosphorylation,whether the SUMOylation of p65 protein is related to the phosphorylated p65,and the link between MANF and phosphorylated p65 have not been reported.Objective: Our purpose isto explore the relationship between SUMO1 and phosphorylated p65,the effects of p65 SUMOylation on p65 phosphorylationand the relationship between MANF and phosphorylated p65.Methods: In HCC patients,the expression of SUMO1 in HCC tissues and paracancer tissues were detected by immunohistochemistry.The expression and localization of SUMO1 in normal liver cell line L02 and hepatoma cell line SMMC-7721 were detected by western blot and immunofluorescence staining.In HCC tissues,immunofluorescence double label assay was used to detect the association of Ki67 and SUMO1.SUMO1 was overexpressed or knocked down in SMMC-7721 cells,and then we used soft agar colony formation assay to observe the effects of SUMO1 on the proliferation of hepatocellular carcinoma cells.In liver tissues of HCC patients,the levels of SUMO1 and phosphorylated p65 protein were detected by immunohistochemistry,and then we used statistical software to analyze the correlation between them.In SMMC-7721 cells,we used WB to dected the effects of overexpression or knockdown of SUMO1 on phosphorylated p65 protein.The effects of MANF on SUMO1 and phosphorylated p65 protein were examined by immunohistochemistry in the tumor-bearing mouse model.The interaction between SUMO1 and MANF was detected by co-immunoprecipitation(Co-IP).In liver cancer tissues,the relationship between MANF and phosphorylated p65 protein were observed by immunofluorescence double labeling.Using the plasmids of GFP-p65 wild-type(GFP-p65-wt)and GFP-p65-mutants plasmids in our laboratory, the target fragments were constructed on the p FLAG-CMV-6a vector by the methods such as enzyme digestion,gel recovery,ligation.Then we expected to use the plasmids for Co-IP experiments to find the SUMO site ofp65.Results: 1.SUMO1 is highly expressed in HCC tissues and hepatoma cells The results of immunohistochemistry showed that the expression of SUMO1 protein in HCC tissuesis significantly higher than that inparacancer tissues.The results of western blot showed that SUMO1 protein expression in hepatoma cell line SMMC-7721 is significantly higher than that in liver cell line L02.Immunofluorescence results showed that the expression of SUMO1 in SMMC-7721 cells was significantly increased compared with L02 cells,and SUMO1 was mainly localized in the nuclei of hepatoma cells.2.SUMO1 promotes the proliferation of hepatoma cells Double-labeled immunofluorescence staining results showed that all the Ki67 positive cells in HCC tissues were SUMO1-positive,indicating thatthere is a relationship between SUMO1 and the proliferation of hepatoma cells.In SMMC-7721 cells,soft agar colony formation assay showed that,overexpression of SUMO1 leads to the cell colonies formation increased,on the contrary,knockdown of SUMO1 leads to cell colonies formation decreased.This indicates that SUMO1 promotes the proliferation of hepatoma cells.3.In livertissues of HCC patients,SUMO1 is positively correlated with phosphorylated p65 Immunohistochemistry staining results showed that SUMO1 and phosphorylated p65 were highly expressed in HCCtissues compared withparacancer tissues,and they mainly located in thenuclei of hepatoma cells.The statistical analysis showed that there was a positive correlation between SUMO1 and phosphorylated p65 in liver tissues of HCC patients.4.In hepatoma cells,SUMO1 promotes p65 protein phosphorylation WB results showed thatthe level of phosphorylated p65 protein was increased after transfection of GFP-SUMO1 plasmid in SMMC-7721 cells.On the contrary,when knocked down of SUMO1,the level of phosphoryled p65 decreased,suggesting that SUMO1 promotes p65 protein phosphorylation.5.SUMO1 is positively correlated with phosphorylated p65 in the tumor-bearing mouse model The expression of SUMO1 and the level of phosphorylated p65 were detected by immunohistochemistry in the tumor-bearing mouse model.The results showed that compared with the NC-sh RNA group,the level of SUMO1 and phosphorylated p65 increased in MANF-sh RNA group which was treated with PBS,while the MANF-sh RNA group which was further treated with recombinant MANF protein,SUMO1 and phosphorylated p65 decreased.It was showed that SUMO1 is associated with phosphorylated p65 protein in a tumor-bearing mouse model.6.MANF is modified by SUMO1 in SMMC-7721 cells In SMMC-7721 cells,the exogenous plasmid of FLAG-MANF was transfected,and then the cells were stimulated with TNFa.Immunoprecipitation experiments showed that MANF is modified by SUMO1.7.MANF protein is associated with phosphorylated p65 in HCC tissues Immunofluorescence double-labeled results showed that there wasa co-localization between MANF and phosphorylated p65 in HCC tissues,and they mainly localized in the nuclei.Theresults showed that MANF is associated withphosphorylated p65.8.FLAG-p65-wt and FLAG-p65-mutants were successfullyconstruted The FLAG-p65-wt and FLAG-p65-mutants plasmids were constructed by using the GFP-p65-wt/mutants plasmids in our laboratory.The p FLAG-CMV-6a vector and the GFP-p65-wt/mutants were digested,and then were processed for agarose gel electrophoresis.The corresponding plasmids were obtained after gel recovery,ligation,and small extraction.The sequencing results showed that the FLAG-p65-wt and FLAG-p65-mutants plasmids were successfully constructed.Conclusions: SUMO1 is highly expressed in liver cancer tissues and hepatoma cells and it promotes the proliferation of liver cancer cells.SUMO1 is positively correlated with p65 phosphorylation in liver tissues of HCC patients.MANF is SUMOylated by SUMO1 and there is a co-localization between MANF and phosphorylated p65 in HCC tissue... |
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