title: Lgr5 Methylation in Cancer Stem Cell Differentiation and Prognosis-Prediction in Colorectal Cancer.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Shasha
authors.initials: S
authors.lastname: Su
authors.affiliation: Institute of liver diseases, Affiliated Hospital of Jining Medical University, Shandong, 273100, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Hong
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Jieqiong
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Kequan
authors.initials: K
authors.lastname: Chen
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Xinying
authors.initials: X
authors.lastname: Wang
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Zhongqiu
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Immunology Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy Place, New York, New York 10029, United States of America.
authors.forename: Cassie
authors.initials: C
authors.lastname: Chang
authors.affiliation: Second affiliated hospital of XingTai medical college, Hebei, 054002, China.
authors.forename: Weihua
authors.initials: W
authors.lastname: Han
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gong
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Haitao
authors.initials: H
authors.lastname: Qin
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.affiliation: Immunology Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, 1 Gustave L. Levy Place, New York, New York 10029, United States of America.
authors.forename: Huabao
authors.initials: H
authors.lastname: Xiong
authors.affiliation: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Peng
mesh_terms: D000328:Adult; D000368:Aged; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics* / Q000401:mortality; D019175:DNA Methylation; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D008297:Male; D008875:Middle Aged; D060787:Neoplasm Grading; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology*; D011379:Prognosis; D011401:Promoter Regions, Genetic; D043562:Receptors, G-Protein-Coupled / Q000235:genetics*
chemical_list: C116884:LGR5 protein, human; D043562:Receptors, G-Protein-Coupled