SUMO Proteins Differentially Regulate The Subcellular Localization And Stability Of P65 And The Interaction Of P65 With MANF In Hepatocellular Carcinoma

Small ubiquitin-related modifier (SUMO) proteins are a family of small proteins which resemble the three-dimensional structure of ubiquitin. SUMO proteins participate in an important post-translational modification called SUMOylation, which promotes SUMO proteins binding to target proteins. SUMOylation is involved in regulation of diverse cellular processes, such as transcriptional regulation, subcellular localization, target proteins stability, and maintenance of genome integrity. Meanwhile, a number of studies have shown that SUMOylation also plays an important role in human disease pathogenesis, especially inflammation-related cancer, such as hepatocellular carcinoma (HCC). p65, one of the most important subunits of NF-κB, is a critical regulator of NF-κB transcriptional activity and associates with the development of HCC. Recently, it has been verified that p65 can be SUMOylated by exogenous SUM03 in HEK293T cells and mouse 3T3 fibroblast cells. However, the reports about the relationship of SUMO proteins and p65 in HCC are limited. Our previous study showed that Mesencephalic Astrocyte-derived Neurotrophic factor (MANF) interacted with p65 in the nucleus and decreased the transciptional activity of NF-κB. Thus, we want to know whether SUMO proteins has effect on the interaction of p65 and MANF.ObjectiveTo investigate how SUMO proteins differentially regulates the subcellular localization and stability of p65 in HCC, and whether SUMO proteins affects the interaction of p65 and MANF.MethodsThe expressions of p65 and SUMO proteins were detected in the liver tissues of HCC patients with HBV infection by using immunohistochemistry. We performed double-labeled immunofluorescence staining and co-immunoprecipitation assay to verify the interactions between p65 and SUMO1 or SUMO2/3. We performed western blot, nuclear and cytoplasmic proteins extraction assay and co-immunoprecipitation assay to detect the protein levels and the subcellular localization of p65 and SUMO and the level of p65 SUMOylation after oxygen-hypoxia deprivation (OGD) and inflammatory factor TNF-a treatment. To study the effect of SUMO proteins on NF-κB-mediated gene activation, double luciferase reporter assays were carried out in hepatoma cells. The biological behavior of hepatoma cells was observed through MTT, colony formation assay and migration assay. Co-immunoprecipitation assay was performed to detect the interaction of p65 and MANF after cells transfected with SUMO1 or SUMO2/3 siRNA.Results:1. Expression of p65 and SUMO proteinsImmunohistochemistry assay showed that only a few cells were SUMO 1-positive in the early stage of hepatitis B, and SUMO1 mainly localized in the nuclei. Meanwhile, SUMO1 was remarkably increased in the liver tumor tissues, compared with that in the adjacent non-tumor tissues. By contrast, SUMO2/3 was remarkable increased the adjacent non-tumor tissues, compared with in the tumor tissues, and was mainly localized in the cytoplasm. Moreover, only a few SUMO2/3-positive cells appeared in the liver tissues of hepatitis B at early stage, where SUMO2/3 was found to localize in both nucleus and cytoplasm of the hepatocytes suffered from slight inflammation. In accordance with SUMO2/3 expression, only a few p65-positive cells appeared in the liver tissues of hepatitis B at early stage. p65 was largely expressed in the non-tumor tissues and mainly localized in cytoplasm, although it was also detectable in the tumor tissues, where it mainly localized in the nuclei.2. p65 can be SUMOylated by SUMO1To verify the correlation between SUMO1 and p65, we performed double-labeled immunofluorescent staining and co-immunoprecipitation assay. Immunofluorescent staining showed that SUMO1 and p65 were co-localized in the nucleus in the liver tumor tissues, HepG2 cells, and SMMC7721 cells. To confirm the interaction between SUMO1 and p65, we co-transfected SMMC7721 cells with myc-Ubc9 and GFP-SUMO1. Co-immunoprecipitation assay showed that SUMO1 was pull down when p65 was immunoprecipitated with anti-p65 antibody. We also detected the high-molecular-weight form of p65 in the hepatoma cells. Meanwhile, the interaction between SUMO1 and p65 and the high-molecular weight forms of p65 and SUMO1 were found in the human liver tumor tissues of HCC patients with HBV infection. These results suggest that SUMO1 interacts with p65, and p65 can be SUMOylated by SUMO1 protein.3. p65 can be SUMOylated by SUMO2/3The similar cytoplasmic characteristics of p65 and SUMO2/3 in live tissues imply an correlation. To confirm it, we performed an immunohistochemical analysis to observe the expressions of p65 and SUMO2/3 in serial sections of HCC. Fortunately, we observed both tumor and non-tumor in a field of microscope. The results showed that the levels of p65 and SUMO2/3 were decreased in tumor tissues, compared with that in the non-tumor. Consistently, the high levels of p65 and SUMO2/3 were found in the same regions of the non-tumor. Double-labeled immunofluorescent staining showed that p65 was co-localized with SUMO2/3 in the cytoplasm of the non-tumor tissues, and p65 was co-localized with SUMO2/3 in hepatoma cell lines. Additionally, co-immunoprecipitation assay showed that there was a weak interaction of p65 with SUM02 and SUM03.4. OGD and TNF-a induced SUMO proteins expression and affected on the level of p65 SUMOylationPost-treated with OGD for 150min, we detected that p65 translocation into the nucleus and the up-regulation of SUMO1 and SUMO2/3 proteins lewels. To test whether inflammatory response affected SUMO proteins expression, we treated SMMC7721 cells with TNF-a (10 ng/ml) for the different time points. The results showed that the nuclear p65 was obviously up-regulated 30 min, other than 8 hrs after TNF-a treatment. Consistently, TNF-a treatment for 30 min caused the cytoplasmic conjugated SUMO2/3 increased, whereas nulcear SUMO2/3, nuclear and cytoplasmic SUMO1 were increased after TNF-a treatment for 8 hours. Co-immunoprecipitation assay showed that TNF-a (10 ng/ml,30 min) treatment enhanced the interaction of p65 with SUMO1, SUMO2, and SUMO3, respectively. However, OGD treatment only enhanced the interaction of p65 with SUMO1 and has no effect on the interaction of p65 with SUMO2/3.5. SUMO1 is involved in the regulation of p65 translocation into the nucleusWe firstly over-expressed SUMO1 in SMMC7721 cells and found SUMO1 proteins did not affect on p65 protein levels. Whereas, overexpression SUMO1 can enhanced hypoxia-induced p65 nuclear import and knockdown endogenous SUMO1 attenuated hypoxia-related p65 nuclear translocation. Additionally, knockdown endogenous SUMO1 decreased TNF-α-induced p65 translocation into the nucleus and overexpression SUMO1 has little effect on TNF-α-induced p65 nuclear transport.6. Knockdown endogenous SUMO1 decreases NF-κB transcriptional activityTo detect whether SUMO1 affects on NF-κB transcriptional activity, we performed a luciferase assay. SUMO1 over-expression significantly reduced NF-κB transcriptional activity induced by TNF-α for 8 hrs, but not for 30 min. Knockdown of endogenous SUMO1 significantly decreased NF-κB transcriptional activity. 7. Effects of SUMO1 on the proliferation and migration of hepatoma cellsMTT assay and colony formation assay showed that knockdown SUMO1 suppressed hepatoma cells proliferation, especially with OGD treatment. Meanwhile, SUMO1 over-expression enhanced the migration of hepatoma cells, and knockdown SUMO1 attenuated the migration ability. In order to search for the relationship of SUMO1 proteins and proliferation of hepatoma cells, we performed a double-labeled immunofluorescent staining in the tumor tissues of HCC patients and found that ki67-positive cells are SUMO1-positive, suggesting that SUMO1 proteins associates with the proliferation of HCC cells.8. SUMO2/3-mediated stability of cytoplasmic p65 is related to inhibition of its degradationWe transfected SMMC7721 cells with the different doses of GFP-SUMO2 orGFP-SUM03, and performed a Western blotting assay to detect the level of p65. The results showed that over-expressed SUM02 and SUM03 increased the levels of p65 in a dose-dependent manner. On the contrary, knockdown of endogenous SUMO2/3 reduced p65 protein level. These results suggest that SUMO2/3 can stabilize p65 protein level. Further nuclear and cytoplasmic extraction assay showed that over-expressed SUM02 and SUM03 just increased the levels of the cytoplasmic p65, but not the nuclear p65. Consistently, knockdown of endogenous SUMO2/3 reduced p65 level in the cytoplasm. These results suggest that SUMO2/3 stabilizes the cytoplasmic p65 level. To figure out the reasons that cause p65 increase in cytoplasm, we investigate the effect of SUMO2/3 on p65 mRNA level. We found that SUMO2/3 neither over-expression nor knockdown affect p65 mRNA level. Next, we performed an immunoprecipitation analysis and found that the ubiquitin-conjugated p65 were reduced along with the increase of SUMO2/3-conjugated p65 induced by TNF-α. These findings suggest that SUMO2/3-mediated stability of cytoplasmic p65 may be via suppressing its degradation mediated by ubiquitin-proteasome pathway.9. Effects of SUMO2/3 proteins on the proliferation and migration of hepatoma cellsMTT assay showed that the over-expressions of SUM02 and SUM03 significantly decreased the proliferation of hepatoma cells. Whereas knockdown SUMO2/3 increased the proliferation. However, SUMO2/3 had little effect on the migration of hepatoma cells.10. SUMO proteins regulates the interaction of p65 and MANFTo test whether SUMO proteins affect the interaction of p65 and MANF, co-immunoprecipitation assay was performed. SMMC7721 cells was transfected with SUMO1 or SUMO2/3 siRNA, NC-siRNA was used as a control.48 hrs post-transfected, the interaction of p65 and MANF was detected by a co-immunoprecipitation assay. The result showed that knockdown of endogenous SUMO1 or SUMO2/3 decreased the interactions of p65 and MANF.ConclusionSUMO proteins were involved in the process from chronic hepatitis to HCC by differentially regulating NF-kB signal pathway. SUMO1 mediates the nuclear transport of p65, while SUMO2/3 as a novel mechanism that regulates the stability of the cytoplasmic p65 through suppressing its ubiquitin-related degradation. Additionally, SUMO proteins regulates the interactions of p65 and MANF. As a result, SUMO proteins may exert an impact on the biological behavior of hepatoma cells. Additionally, SUMO proteins were involved in the regulation of the interaction of p65 and MANF.