Su Shasha, Hong Feng, Liang Yanling, Zhou Jieqiong, Liang Yan, Chen Kequan, Wang Xinying, Wang Zhongqiu, Wang Zhiqing, Chang Cassie, Han Weihua, Gong Wei, Qin Haitao, Jiang Bo, Xiong Huabao, Peng Liang
Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
Institute of liver diseases, Affiliated Hospital of Jining Medical University, Shandong, 273100, China.
PLoS One. 2015 Nov 24;10(11):e0143513. doi: 10.1371/journal.pone.0143513. eCollection 2015.
Leucine-rich-repeat-containing G-protein-coupled receptor 5 (lgr5) is a candidate marker for colorectal cancer stem cells (CSC). In the current study, we investigated the methylation status within thelgr5 promoter and evaluated its relationship with CSC differentiation, prognosis for colorectal cancer, and its clinicopathological features.
The methylation status within Lgr5 promoter was detected with a methylation-specific PCR in six colorectal cancer cell lines as well as 169 primary colorectal tumor tissues. Differentiation of CSC was examined with immunofluorescence and immunocytochemistry. Down-regulation of lgr5 was achieved with gene-specific siRNA. The associations between lgr5 methylation and the clinicopathological features as well as survival of patients were analyzed with statistical methods.
The lgr5 promoter was methylated to different degrees for the six colorectal cell lines examined, with complete methylation observed in HCT116 cells in which the lgr5 expression was partially recovered following DAC treatment. The stem-cell sphere formation from HCT116 cells was accompanied by increasing methylation within the lgr5 promoter and decreasing expression of lgr5. Knocking down lgr5 by siRNA also led to stem-cell spheres formation. Among primary colorectal tumors, 40% (67/169) were positive for lgr5 methylation, while none of the normal colon tissues were positive for lgr5 methylation. Furthermore, lgr5 methylation significantly associated with higher tumor grade, and negative distant metastasis (p < 0.05), as well as better prognosis (p = 0.001) in patients with colorectal cancer.
Our data suggests that lgr5 methylation, through the regulation of lgr5 expression and colorectal CSC differentiation, may constitute a novel prognostic marker for colorectal cancer patients.
富含亮氨酸重复序列的G蛋白偶联受体5（lgr5）是结直肠癌干细胞（CSC）的候选标志物。在本研究中，我们调查了lgr5启动子内的甲基化状态，并评估了其与CSC分化、结直肠癌预后及其临床病理特征的关系。
采用甲基化特异性PCR检测6种结直肠癌细胞系以及169例原发性结直肠肿瘤组织中Lgr5启动子的甲基化状态。通过免疫荧光和免疫细胞化学检测CSC的分化情况。用基因特异性小干扰RNA（siRNA）实现lgr5的下调。采用统计学方法分析lgr5甲基化与临床病理特征以及患者生存之间的关联。
在所检测的6种结直肠癌细胞系中，lgr5启动子呈现不同程度的甲基化，在HCT116细胞中观察到完全甲基化，用5-氮杂-2'-脱氧胞苷（DAC）处理后lgr5表达部分恢复。HCT116细胞形成干细胞球伴随着lgr5启动子内甲基化增加和lgr5表达降低。用siRNA敲低lgr5也导致干细胞球形成。在原发性结直肠肿瘤中，40%（67/169）的lgr5甲基化呈阳性，而正常结肠组织中lgr5甲基化均为阴性。此外，lgr5甲基化与结直肠癌患者的肿瘤分级较高、远处转移阴性（p<0.05）以及预后较好（p=0.001）显著相关。
我们的数据表明，lgr5甲基化通过调节lgr5表达和结直肠CSC分化，可能构成结直肠癌患者的一种新的预后标志物。
