College of Life Science and Bioengineering, Beijing University of Technology, Beijing, 100124, China.
Department of Gastroenterology and Hepatology, Chinese PLA General Hospital, #28 Fuxing Road, Beijing, 100853, China.
Clin Epigenetics. 2018 Nov 6;10(1):137. doi: 10.1186/s13148-018-0570-4.
The role of TMEM176A in human hepatocellular carcinoma (HCC) is unknown. This study explored the epigenetic regulation and function of TMEM176A in human HCC.
Twelve HCC cell lines and 126 cases of primary cancer were analyzed. Methylation-specific PCR, immunohistochemistry, flow cytometry, and xenograft mouse models were employed.
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.
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.
TMEM176A 在人肝细胞癌（HCC）中的作用尚不清楚。本研究旨在探讨 TMEM176A 在人 HCC 中的表观遗传调控和功能。
分析了 12 个人肝癌细胞系和 126 例原发性肝癌组织。采用甲基化特异性 PCR、免疫组织化学、流式细胞术和异种移植小鼠模型进行研究。
在 SNU387、SNU182、Huh1 和 SNU475 细胞中，TMEM176A 高表达；在 HepG2 和 PLC/PRF/5 细胞中，TMEM176A 低表达；在 SNU449、HBXF344、SMMC7721、Huh7 和 LM3 细胞中则无表达。在 SNU387、SNU182、Huh1 和 SNU475 细胞中，TMEM176A 启动子呈非甲基化状态；在 HepG2 和 PLC/PRF/5 细胞中，TMEM176A 启动子呈部分甲基化状态；在 SNU449、HBXF344、SMMC7721、Huh7 和 LM3 细胞中，TMEM176A 启动子呈完全甲基化状态。用 5-氮杂-2′-脱氧胞苷处理后，SNU449、HBXF344、SMMC7721、Huh7 和 LM3 细胞中 TMEM176A 的表达重新出现；HepG2 和 PLC/PRF/5 细胞中 TMEM176A 的表达增加；而 SNU387、SNU182、Huh1 和 SNU475 细胞中的 TMEM176A 表达则无变化。在 126 例原发性 HCC 中，TMEM176A 启动子区有 75.4%（95/126）发生甲基化。TMEM176A 表达降低与启动子区甲基化有关（P<0.05）。TMEM176A 启动子甲基化与年龄、性别、HBV 感染、肝硬化、肿瘤大小、淋巴结转移、血管癌栓、病灶数量和 TNM 分期均无相关性（均 P>0.05）。这些结果表明，TMEM176A 的表达受启动子区甲基化的调控。TMEM176A 启动子的甲基化与肿瘤细胞分化显著相关（P<0.05），是 3 年总生存预后不良的独立预测因素（P<0.05）。TMEM176A 表达诱导细胞凋亡；抑制细胞增殖、迁移和侵袭；抑制人 HCC 细胞异种移植在小鼠体内的生长；并抑制 HCC 细胞中的 ERK 信号通路。
TMEM176A 启动子在人 HCC 中经常发生甲基化，TMEM176A 的表达受启动子区甲基化的调控。TMEM176A 启动子甲基化可能是 HCC 的诊断和预后标志物。TMEM176A 通过抑制 ERK 信号通路抑制 HCC 的生长。
