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. 2015 Oct 12:15:675.
doi: 10.1186/s12885-015-1665-3.

Small ubiquitin-related modifier 2/3 interacts with p65 and stabilizes it in the cytoplasm in HBV-associated hepatocellular carcinoma

Jun Liu  1   2 , Manqi Sha  3   4 , Qianfeng Wang  5   6 , Yong Ma  7 , Xiaoping Geng  8 , Yufeng Gao  9 , Lijie Feng  10   11 , Yujun Shen  12   13 , Yuxian Shen  14   15
Affiliations

Small ubiquitin-related modifier 2/3 interacts with p65 and stabilizes it in the cytoplasm in HBV-associated hepatocellular carcinoma

Jun Liu et al. BMC Cancer. .

Abstract

Background: SUMOylation, an important post-translational modification, associates with the development of hepatocellular carcinoma (HCC). p65, one of the most important subunits of NF-κB, is a key regulator in the development of HCC and has been reported to be SUMOylated by exogenous small ubiquitin-related modifier 3 (SUMO3) in HEK 293T cells. However, the relationship between p65 and SUMO2/3 in HCC remains unknown. This study was to investigate the interaction between p65 and SUMO2/3 and explore the potential roles involved in HCC.

Methods: The expressions of p65 and SUMO2/3 in the liver tissues were detected by using immunohistochemistry. We performed double-labeled immunofluorescence and co-immunoprecipitation assay to verify the interaction between p65 and SUMO2/3. The extraction of nuclear and cytoplasmic proteins was performed, and the subcellular localization of p65 was detected. The proliferation and migration of hepatoma cells were observed using MTT, colony formation, and transwell assays.

Results: We found a strong SUMO2/3-positive immunoreactivity in the cytoplasm in the non-tumor tissues of HCC. However, SUMO2/3 level was down regulated in the tumor tissues as compared with the adjacent non-tumor tissues. In accordance with this finding, p65 was up regulated in the adjacent non-tumor tissues and almost localized in the cytoplasm. There was a close correlation between SUMO2/3 and p65 expressions in the liver tissues (R = 0.800, p = 0.006). The interaction between p65 and SUMO2/3 was verified by co-immunoprecipitation and double-labeled immunofluorescent assays. TNF-α (10 ng/ml) treatment for 30 min not only up regulated the cytoplasmic conjugated SUMO2/3, but also enhanced SUMO2/3-p65 interaction. Furthermore, we found that SUMO2/3 up regulated the cytoplasmic p65 protein level in a dose-dependent manner, but not affected its mRNA level. The increase of p65 protein by SUMO2/3 was abolished by MG132 treatment, a reversible inhibitor of proteasome. Meanwhile, TNF-α-induced increase of SUMO2/3-conjugated p65 was along with the reduction of the ubiquitin-conjugated p65. The further study showed that SUMO2/3 over-expression decreased the proliferative ability of hepatoma cells, but did not affect the migration.

Conclusion: SUMO2/3-p65 interaction may be a novel mechanism involved in the transformation from chronic hepatitis B to HCC via stabilizing cytoplasmic p65, which might shed light on understanding the tumorigenesis and development.

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Figures

Fig. 1
Fig. 1
The profile of SUMO2/3 expression in the liver tissues. SUMO2/3 expression was detected using immunohistochemistry in the liver tissues of hepatitis B (d-f), HCC (g-i), and corresponding adjacent non-tumor tissues (j-l). a-c The isotype IgG control. b, e, h, and k are the magnified images of the rectangles in a , d, g, and j, respectively. c, f, i, and l are the magnified images of the rectangles in b, e, h, and k, respectively. The scale bars were shown as indicated. m The integral optical density (IOD) in panel g-l. The data were expressed as the means ± SD of the 5 individuals (n = 5). *p < 0.05, compared with Pa. Ca: tumor; Pa: para-tumor. n The levels of SUMO2/3 were detected by western blot. o The quantitative data in panel n. The data were expressed as the means ± SD of the 4 individuals (n = 4). **p < 0.01, compared with Pa. Ca: tumor; Pa: para-tumor
Fig. 2
Fig. 2
The profile of p65 expression in the liver tissues. p65 expression was detected using immunohistochemistry in the liver tissues of hepatitis B (d-f), HCC (g-i), and corresponding adjacent non-tumor tissues (j-l). a-c The isotype IgG control. b, e, h, and k are the magnified images of the rectangles in a, d, g, and j, respectively. c, f, i, and l are the magnified images of the rectangles in b, e, h, and k, respectively. The scale bars were shown as indicated. m The integral optical density (IOD) in panel g-l. The data were expressed as the means ± SD of the 8 individuals (n = 8). *p < 0.05, compared with Pa. Ca: tumor; Pa: para-tumor. n The levels of p65 were detected by western blot. o The quantitative data in panel n. The data were expressed as the means ± SD of the 4 individuals (n = 4). **p < 0.01, compared with Pa. Ca: tumor; Pa: para-tumor. p Pearson’s correlation test was used to analyse the relationship between the IODs of SUMO2/3 and p65 in the human liver tissues. p = 0.006, n = 9, R = 0.800
Fig. 3
Fig. 3
SUMO2/3 interacts with p65 in liver tissues and hepatoma cell lines. a The similar profiles of p65 and SUMO2/3 in the liver tissues. p65 and SUMO2/3 were detected using immunohistochemistry in the serial sections of the liver tissues. The images of the rectangles in the upper panels are magnified in the lower panel. The scale bars were shown as indicated. The arrows indicate the tumor tissues. b-d Co-localization of SUMO2/3 and p65. Double-labeled immunofluorescent staining was performed using the antibodies against p65 (green) and SUMO2/3 (red) in the liver tissues (b), HepG2 cells (c) and SMMC7721 cells (d). The nuclei were stained with DAPI (blue). In panel b, the images of the rectangles in the upper panels are magnified in the lower panels. The scale bars were shown as indicated. e-f Interaction of p65 and SUMO2/3 in the hepatoma cells. SMMC7721 cells were co-transfected with myc-Ubc9 and GFP-SUMO2 (e) or GFP-SUMO3 (f). At 24 h after transfection, the cells were lyzed and incubated with Protein A agarose containing anti-p65 antibody. g Interaction of p65 and SUMO2/3 in the liver tissues. The non-tumor tissues collected from HCC patients were lyzed and co-immunoprecipitation assay was performed using anti-p65 antibody. The isotype IgG was used as a negative control. The bound proteins were blotted by using the antibodies as indicated. 2 % total lysate was loaded as input
Fig. 4
Fig. 4
TNF-α up-regulates SUMO2/3 and promotes p65 SUMOylation. a-b SMMC7721 cells were treated with TNF-α (10 ng/ml) for 30 min (a) and 8 h (b), respectively. The nuclear (N) and cytoplasmic (C) fragments were extracted by using the protein extraction kit according to the manufacturer’s instruction. The proteins were blotted with the antibodies as indicated. Tubulin and histone H3 were used as the markers of cytoplasm and nucleus, respectively. c-d SMMC7721 cells were co-transfected with myc-Ubc9 and GFP-SUMO2 (c) or GFP-SUMO3 (d). At 24 h after transfection, the cells were treated with TNF-α (10 ng/ml) for 30 min. Then the cells were lyzed and incubated with anti-p65 antibody. The isotype IgG was used as a negative control. The bound proteins were blotted by using the antibodies as indicated. 2 % total lysate was loaded as input
Fig. 5
Fig. 5
SUMO2/3 stabilizes the cytoplasmic p65. a-b Dose-dependent effects of SUMO2/3 on p65 level. SMMC7721 cells were transfected with GFP-SUMO2 (a) or GFP-SUMO3 (b). The blank vector (pEGFP-C1) was transfected to balance the total DNA. At 24 h after transfection, the cells were collected and processed for immunoblotting. The data were expressed as the means ± SD of at least three independent experiments. **p < 0.01, ***p < 0.001, compared with pEGFP-1 vector. c SUMO2/3 knockdown decreased p65 level. SMMC7721 cells were transfected with SUMO2/3-siRNA (siRNA) or negative control siRNA (control). Western blotting assay was performed 48 h after transfection. The data were expressed as the means ± SD of at least three independent experiments. *p < 0.05, compared with the control. d-e SUMO2/3 increased the cytoplasmic p65. SMMC7721 cells were transfected with GFP-SUMO2 (d) or GFP-SUMO3 (e). The blank vector (pEGFP-C1) was transfected to balance the total DNA. Nuclear and cytoplasmic proteins were extracted by using the protein extraction kit according to the manufacturer’s instructions. Tubulin and histone H3 were used as the markers of cytoplasm and nucleus, respectively. f SUMO2/3 knockdown decreased the cytoplasmic p65. SMMC7721 cells were transfected with SUMO2/3-siRNA. NC (negative control) siRNA was used as a control. Nuclear and cytoplasmic proteins were extracted 48 h after transfection and processed for Western blotting
Fig. 6
Fig. 6
Effect of SUMO2/3 on p65 mRNA expression. SMMC7721 cells were transfected with the plasmids or siRNA as indicated. The expressions of SUMO2/3 proteins and p65 mRNA were detected using Western blotting and RT-PCR, respectively (a). The relative mRNA was quantitative analyzed in (b). The data were expressed as the means ± SD of at least three independent experiments
Fig. 7
Fig. 7
Proteasome inhibitor MG132 abolished SUMO2/3-mediated p65 stability. SMMC7721 cells were transfected with GFP-SUMO2 (a) and GFP-SUMO3 (b). At 24 h after transfection, the cells were treated with MG132 (20 μM) for 6 h. Then the lysate was subjected to Western blotting analysis
Fig. 8
Fig. 8
Effects of SUMO2/3 on the proliferation and migration of hepatoma cells. SMMC7721 cells were transfected with SUMO2/3 plasmid or SUMO2/3-siRNA. At 48 h after transfection, Western blot was used to detect SUMO2/3 expression (a). The proliferation was determined using MTT assay (b) and colony formation assay (c-d). The migration was detected using transwell assay (e-f). The data were expressed as the means ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with pEGFP-C1 vector control
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References

    1. Martin S, Wilkinson KA, Nishimune A, Henley JM. Emerging extranuclear roles of protein SUMOylation in neuronal function and dysfunction. Nat Rev Neurosci. 2007;8:948–959. doi: 10.1038/nrn2276. - DOI - PMC - PubMed
    1. Guo D, Li M, Zhang Y, Yang P, Eckenrode S, Hopkins D, Zheng W, Purohit S, Podolsky RH, Muir A, Wang J, Dong Z, Brusko T, Atkinson M, Pozzilli P, Zeidler A, Raffel LJ, Jacob CO, Park Y, Serrano-Rios M, Larrad MT, Zhang Z, Garchon HJ, Bach JF, Rotter JI, She JX, Wang CY. A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes. Nat Genet. 2004;36:837–841. doi: 10.1038/ng1391. - DOI - PubMed
    1. Flotho A, Melchior F. Sumoylation: a regulatory protein modification in health and disease. Annu Rev Biochem. 2013;82:357–385. doi: 10.1146/annurev-biochem-061909-093311. - DOI - PubMed
    1. Johnson ES. Protein modification by SUMO. Annu Rev Biochem. 2004;73:355–382. doi: 10.1146/annurev.biochem.73.011303.074118. - DOI - PubMed
    1. Dasso M. Biochemistry: Rear view of an enzyme. Nature. 2013;497:576–577. doi: 10.1038/nature12249. - DOI - PubMed

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