title: Increased levels of the long intergenic non-protein coding RNA POU3F3 promote DNA methylation in esophageal squamous cell carcinoma cells.
abstract: OBJECTIVE
  Thousands of long intergenic non-protein coding RNAs (lincRNAs) have been identified in mammals via genome-wide sequencing studies. Many are functional, but are expressed aberrantly by cancer cells. We investigated whether levels of lincRNAs are altered during the development of esophageal squamous cell carcinoma (ESCC).
  
  
  METHODS
  We used quantitative real-time polymerase chain reaction to measure levels of 26 highly conserved lincRNAs in ESCC and surrounding nontumor tissues. A total of 182 ESCC and paired adjacent nontumor tissue samples were collected from patients undergoing tylectomy at The First Affiliate Hospital of Soochow University from 2001 through 2009; another 178 ESCC tissue pairs were collected from Guangzhou Medical University from 2002 through 2009. LincRNAs were expressed from lentiviral vectors or knocked down with small hairpin RNAs in Eca-109 and TE-1 cells.
  
  
  RESULTS
  Levels of a lincRNA encoded by a gene located next to POU3F3 (linc-POU3F3) were significantly higher in ESCC than neighboring nontumor tissues. In RNA immunoprecipitation assays, linc-POU3F3 was associated with the EZH2 messenger RNA (mRNA). Overexpression of linc-POU3F3 in cell lines increased their proliferation and ability to form colonies, and reduced the expression of POU3F3 mRNA, whereas knockdown of linc-POU3F3 increased the levels of POU3F3 mRNA. CpG islands in POU3F3 were densely hypermethylated in cell lines that overexpressed linc-POU3F3; methylation at these sites was reduced by knockdown of linc-POU3F3. Pharmacologic inhibition of EZH2 increased the levels of POU3F3 mRNA and significantly reduced binding of DNA methyltransferase (DNMT)1, DNMT3A, and DNMT3B to POU3F3. ESCC cells with knockdown of linc-POU3F3 formed xenograft tumors more slowly in mice than control ESCC cells.
  
  
  CONCLUSIONS
  Levels of linc-POU3F3 are increased in ESCC samples from patients compared with nontumor tissues. This noncoding RNA contributes to the development of ESCC by interacting with EZH2 to promote methylation of POU3F3, which encodes a transcription factor.
authors.affiliation: Department of Genetics, Medical College of Soochow University, Suzhou, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Genetics, Medical College of Soochow University, Suzhou, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Department of Genetics, Medical College of Soochow University, Suzhou, China.
authors.forename: Jieqiong
authors.initials: J
authors.lastname: Deng
authors.affiliation: Department of Genetics, Medical College of Soochow University, Suzhou, China.
authors.forename: Yonghe
authors.initials: Y
authors.lastname: You
authors.affiliation: Department of Genetics, Medical College of Soochow University, Suzhou, China.
authors.forename: Hongchun
authors.initials: H
authors.lastname: Wu
authors.affiliation: Department of Genetics, Medical College of Soochow University, Suzhou, China.
authors.forename: Na
authors.initials: N
authors.lastname: Li
authors.affiliation: The Institute for Chemical Carcinogenesis, The State Key Lab of Respiratory Disease, Guangzhou Medical University, Guangzhou, China.
authors.forename: Jiachun
authors.initials: J
authors.lastname: Lu
authors.affiliation: Department of Genetics, Medical College of Soochow University, Suzhou, China. Electronic address: zhouyifeng@suda.edu.cn.
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Zhou
mesh_terms: D000328:Adult; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000601:surgery; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002681:China; D018899:CpG Islands; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D000090205:DNA Methyltransferase 3A; D000071221:Enhancer of Zeste Homolog 2 Protein; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000601:surgery; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D008969:Molecular Sequence Data; D050809:POU Domain Factors / Q000235:genetics / Q000378:metabolism*; D063151:Polycomb Repressive Complex 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D047368:Tumor Burden; D015854:Up-Regulation; D000097584:DNA Methyltransferase 3B
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D000071221:Enhancer of Zeste Homolog 2 Protein; D004791:Enzyme Inhibitors; D050809:POU Domain Factors; D063151:Polycomb Repressive Complex 2; D062085:RNA, Long Noncoding; D012333:RNA, Messenger; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; C000589675:POU3F3 protein, human; C498409:EZH2 protein, human
keywords: Epigenetics; Esophageal Cancer; Gene Regulation; Tumor Suppressor