Gao Dan, Han Yingjie, Yang Yang, Herman James G, Linghu Enqiang, Zhan Qimin, Fuks François, Lu Zhi John, Guo Mingzhou
a Department of Gastroenterology & Hepatology , Chinese PLA General Hospital , Beijing , China.
b School of Medicine, Nankai University , Tianjin , China.
Epigenetics. 2017 Jul 3;12(7):575-583. doi: 10.1080/15592294.2017.1341027. Epub 2017 Jul 5.
Colorectal cancer (CRC) is the third most common malignancy and the fourth most common cause of cancer related death worldwide. This study was designed to find tumor suppressors involved in CRC development by performing RNA-seq. Eight CRC cell lines and 130 cases of primary CRC samples were used. RNA-seq, methylation-specific PCR (MSP), flow cytometry, transwell assays, and a xenograft mouse model were used. Reduction of TMEM176A expression was confirmed in human CRC cells by RNA-seq. TMEM176A was expressed in LS180 and SW620 cells, loss of TMEM176A expression was observed in LOVO, HCT116, RKO, and DLD1 cells, and reduced TMEM176A expression was found in HT29 and SW480 cells. Unmethylation of the TMEM176A promoter was found in LS180 and SW620 cells, whereas complete methylation was found in LOVO, HCT116, RKO, and DLD1 cells, and partial methylation was found in HT29 and SW480 cells. Promoter region methylation correlated with loss of/reduced expression of TMEM176A. Re-expression of TMEM176A was induced by 5-aza-2'-deoxycytidine. TMEM176A was methylated in 50.77% of primary colorectal cancers. Methylation of TMEM176A was associated with tumor metastasis (P<0.05) and was an independent prognostic factor for 5-year overall survival (OS) according to Cox proportional hazards model analysis (P<0.05). TMEM176A induced apoptosis and inhibited cell migration and invasion in CRC cells. TMEM176A suppressed CRC cell growth both in vitro and in vivo. Our results suggest that expression of TMEM176A is regulated by promoter region methylation. TMEM176A methylation is an independent prognostic marker for 5-year OS in CRC, and may act as a tumor suppressor in CRC.
结直肠癌（CRC）是全球第三大常见恶性肿瘤，也是癌症相关死亡的第四大常见原因。本研究旨在通过RNA测序寻找参与结直肠癌发生发展的肿瘤抑制因子。使用了8种结直肠癌细胞系和130例原发性结直肠癌样本。采用了RNA测序、甲基化特异性PCR（MSP）、流式细胞术、Transwell实验和异种移植小鼠模型。通过RNA测序证实人结直肠癌细胞中TMEM176A表达降低。TMEM176A在LS180和SW620细胞中表达，在LOVO、HCT116、RKO和DLD1细胞中观察到TMEM176A表达缺失，在HT29和SW480细胞中发现TMEM176A表达降低。在LS180和SW620细胞中发现TMEM176A启动子未甲基化，而在LOVO、HCT116、RKO和DLD1细胞中发现完全甲基化，在HT29和SW480细胞中发现部分甲基化。启动子区域甲基化与TMEM176A表达缺失/降低相关。5-氮杂-2'-脱氧胞苷诱导TMEM176A重新表达。50.77%的原发性结直肠癌中TMEM176A发生甲基化。TMEM176A甲基化与肿瘤转移相关（P<0.05），根据Cox比例风险模型分析，是5年总生存期（OS）的独立预后因素（P<0.05）。TMEM176A诱导结直肠癌细胞凋亡并抑制细胞迁移和侵袭。TMEM176A在体外和体内均抑制结直肠癌细胞生长。我们的结果表明，TMEM176A的表达受启动子区域甲基化调控。TMEM176A甲基化是结直肠癌5年OS的独立预后标志物，可能在结直肠癌中起肿瘤抑制作用。
