title: Small ubiquitin-related modifier 2/3 interacts with p65 and stabilizes it in the cytoplasm in HBV-associated hepatocellular carcinoma.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: School of Pharmacy, Anhui Medical University, 81 Meishan Road, Hefei, 230032, PR China. liujun841030@aliyun.com.|Biopharmaceutical Research Institute, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, PR China. liujun841030@aliyun.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.affiliation: School of Pharmacy, Anhui Medical University, 81 Meishan Road, Hefei, 230032, PR China. 1150546037@qq.com.|Biopharmaceutical Research Institute, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, PR China. 1150546037@qq.com.
authors.forename: Manqi
authors.initials: M
authors.lastname: Sha
authors.affiliation: Biopharmaceutical Research Institute, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, PR China. 799044364@qq.com.|School of Basic Medical Sciences, Anhui Medical University, 81 Meishan Road, Hefei, 230032, PR China. 799044364@qq.com.
authors.forename: Qianfeng
authors.initials: Q
authors.lastname: Wang
authors.affiliation: The Chinese People's Liberation Army 123 Hospital, 1052 Yanshan Road, Bengbu, 233015, PR China. 921999181@qq.com.
authors.forename: Yong
authors.initials: Y
authors.lastname: Ma
authors.affiliation: The First Affiliated Hospital, Anhui Medical University, 218 Jixi Road, Hefei, 230022, PR China. gengwwl2004@163.com.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Geng
authors.affiliation: The Second Affiliated Hospital, Anhui Medical University, 678 Furong Road, Hefei, 230601, PR China. aygyf@126.com.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Biopharmaceutical Research Institute, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, PR China. fenglijie1128@sina.com.|School of Basic Medical Sciences, Anhui Medical University, 81 Meishan Road, Hefei, 230032, PR China. fenglijie1128@sina.com.
authors.forename: Lijie
authors.initials: L
authors.lastname: Feng
authors.affiliation: Biopharmaceutical Research Institute, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, PR China. jojo125520@163.com.|School of Basic Medical Sciences, Anhui Medical University, 81 Meishan Road, Hefei, 230032, PR China. jojo125520@163.com.
authors.forename: Yujun
authors.initials: Y
authors.lastname: Shen
authors.affiliation: Biopharmaceutical Research Institute, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, PR China. shenyx@ahmu.edu.cn.|School of Basic Medical Sciences, Anhui Medical University, 81 Meishan Road, Hefei, 230032, PR China. shenyx@ahmu.edu.cn.
authors.forename: Yuxian
authors.initials: Y
authors.lastname: Shen
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000209:etiology* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D003593:Cytoplasm / Q000378:metabolism; D015870:Gene Expression; D006509:Hepatitis B / Q000150:complications*; D006801:Humans; D008113:Liver Neoplasms / Q000209:etiology* / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding; D055550:Protein Stability; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D051996:Transcription Factor RelA / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: C117909:SUMO2 protein, human; C492189:SUMO3 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D014452:Ubiquitins