Epigenetic silencing of TMEM176A activates ERK signaling in human hepatocellular carcinoma

Clin Epigenetics. 2018 Nov 6;10(1):137. doi: 10.1186/s13148-018-0570-4.

Abstract

Background: The role of TMEM176A in human hepatocellular carcinoma (HCC) is unknown. This study explored the epigenetic regulation and function of TMEM176A in human HCC.

Materials and methods: Twelve HCC cell lines and 126 cases of primary cancer were analyzed. Methylation-specific PCR, immunohistochemistry, flow cytometry, and xenograft mouse models were employed.

Results: TMEM176A was highly expressed in SNU387, SNU182, Huh1, and SNU475 cells; reduced expression was observed in HepG2 and PLC/PRF/5 cells; and no expression was found in SNU449, HBXF344, SMMC7721, Huh7, and LM3 cells. Unmethylation of the TMEM176A promoter was detected in SNU387, SNU182, Huh1, and SNU475 cells; partial methylation was observed in HepG2 and PLC/PRF/5 cells; and complete methylation was found in SNU449, HBXF344, SMMC7721, Huh7, and LM3 cells. Upon treatment with 5-Aza-2-deoxycytidine, re-expression of TMEM176A was detected in SNU449, HBXF344, SMMC7721, Huh7, and LM3 cells; increased expression of TMEM176A was observed in HepG2 and PLC/PRF/5 cells; and no expression changes were found in SNU387, SNU182, Huh1, and SNU475 cells. The TMEM176A promoter region was methylated in 75.4% (95/126) of primary human HCC. Reduced expression of TMEM176A was associated with promoter region methylation (P < 0.05). No association was found between TMEM176A promoter methylation and age, gender, HBV infection, liver cirrhosis, tumor size, lymph node metastasis, vessel cancerous embolus, number of lesions, and TNM stage (all P > 0.05). These results demonstrated that the expression of TMEM176A is regulated by promoter region methylation. Methylation of the TMEM176A promoter was significantly associated with tumor cell differentiation (P < 0.05) and was an independent prognostic factor for poor 3-year overall survival (OS, P < 0.05). TMEM176A expression induced cell apoptosis; inhibited cell proliferation, migration, and invasion; suppressed human HCC cell xenograft growth in mice; and inhibited ERK signaling in HCC cells.

Conclusion: The promoter region of TMEM176A is frequently methylated in human HCC, and the expression of TMEM176A is regulated by promoter region methylation. Methylation of the TMEM176A promoter may serve as a diagnostic and prognostic marker in HCC. TMEM176A suppresses HCC growth by inhibiting the ERK signaling pathway.

Keywords: DNA methylation; ERK1/2; HCC; SAR1A; TMEM176A.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Animals
  • Carcinoma, Hepatocellular / genetics
  • Carcinoma, Hepatocellular / pathology*
  • Cell Line, Tumor
  • Cell Movement
  • Cell Proliferation
  • DNA Methylation*
  • Epigenesis, Genetic
  • Female
  • Gene Expression Regulation, Neoplastic
  • Hep G2 Cells
  • Humans
  • Liver Neoplasms / genetics
  • Liver Neoplasms / pathology*
  • MAP Kinase Signaling System
  • Male
  • Membrane Proteins / genetics*
  • Mice
  • Middle Aged
  • Neoplasm Transplantation
  • Promoter Regions, Genetic
  • Up-Regulation*

Substances

  • Membrane Proteins
  • TMEM176A protein, human