Increased levels of the long intergenic non-protein coding RNA POU3F3 promote DNA methylation in esophageal squamous cell carcinoma cells.

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). 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. 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. 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.

Li W ，Zheng J ，Deng J ，You Y ，Wu H ，Li N ，Lu J ，Zhou Y ... -  《-》

Functional linc-POU3F3 is overexpressed and contributes to tumorigenesis in glioma.

Growing number of long intergenic noncoding RNAs (lincRNAs) have recently been identified in mammals as new modulators in cancer origination and progression involved in a broad range of biological processes. Long intergenic noncoding RNA POU3F3 (linc-POU3F3) has been characterized as a highly conserved functional transcription regulator in esophageal squamous cell carcinoma. The contributions of this lincRNA to glioblastoma remain unknown. In this present study, we investigated the expression pattern and functional role of linc-POU3F3 in glioma by using real-time PCR and gain-/loss-of-function studies. The results revealed that linc-POU3F3 levels were extraordinarily associated with the tumor WHO grade. In related biochemical assays, overexpression of linc-POU3F3 promotes cell viability and proliferation in glioma cells, whereas knockdown of linc-POU3F3 showed the opposite effect. As expected, we also found that linc-POU3F3 expression was negatively correlated with the mRNA level of POU3F3 (the evolutionarily conserved neighbor gene of linc-POU3F3). Our results indicate that linc-POU3F3 might affect glioma development via altering expression level of POU3F3, and lead us to believe that linc-POU3F3 may also have a crucial regulatory role in glioma progression.

Guo H ，Wu L ，Yang Q ，Ye M ，Zhu X ... -  《-》

Increased expression of EIF5A2, via hypoxia or gene amplification, contributes to metastasis and angiogenesis of esophageal squamous cell carcinoma.

Solid tumors often become hypoxic, leading to activation of hypoxia-response genes. We investigated the effects of overexpression of the hypoxia response genes eIF5A2 in esophageal squamous cell carcinoma (ESCC). We used quantitative real-time polymerase chain reaction and immunohistochemistry analyses to compare expression of eIF5A2 between paired ESCC samples and nontumor esophageal tissues, and fluorescence in situ hybridization to detect gene copy-number alterations. Luciferase reporter and chromatin immunoprecipitation assays were used to study interactions between eIF5A2 and hypoxia-inducible factor-1α (HIF1α). We determined the effects of eIF5A2 overexpression and knockdown in ESCC cell lines and growth of ESCC xenograft tumors in nude mice. Levels of eIF5A2 messenger RNA and protein were increased in >40% of ESCC samples compared with matched nontumor tissues, along with levels of HIF1α and vascular endothelial growth factor. Increased levels of EIF5A2 were significantly associated with ESCC metastasis to lymph nodes (P < .001) and tissue invasion (P = .037), and shorter survival times of patients (P < .001). Amplification of eIF5A2 was detected in 35.14% of ESCC samples that overexpressed eIF5A2. Hypoxia increased expression of eIF5A2 4- to 8-fold in ESCC cell lines; we observed bidirectional regulation between eIF5A2 and HIF1α. Transient transfection of ESCC cell lines with eIF5A2 increased their migratory and invasive abilities and markers of the epithelial to mesenchymal transition, and eIF5A2 knockdown or HIFα inhibition reduced these. In mice, xenograft tumors grown from ESCC cells that expressed eIF5A2 formed tumors more rapidly than cells that expressed only vector (controls); they also expressed higher levels of HIF1α and vascular endothelial growth factor, and formed more microvessels than controls. Knockdown of eIF5A2 in ESCC cells with interfering RNAs reduced their growth as xenograft tumors in mice, particularly when mice were given docetaxel or cisplatin. eIF5A2 is overexpressed by gene amplification or hypoxia in ESCCs, and associated with up-regulation of HIF1α, metastasis, and shorter survival times of patients. Increased expression of eIF5A2 increases metastasis and angiogenesis in ESCC via the HIF1α-mediated signaling pathway.

Li Y ，Fu L ，Li JB ，Qin Y ，Zeng TT ，Zhou J ，Zeng ZL ，Chen J ，Cao TT ，Ban X ，Qian C ，Cai Z ，Xie D ，Huang P ，Guan XY ... -  《-》

Identification of PTK6, via RNA sequencing analysis, as a suppressor of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most commonly observed histologic subtype of esophageal cancer. ESCC is believed to develop via accumulation of numerous genetic alterations, including inactivation of tumor suppressor genes and activation of oncogenes. We searched for transcripts that were altered in human ESCC samples compared with nontumor tissues. We performed integrative transcriptome sequencing (RNA-Seq) analysis using ESCC samples from 3 patients and adjacent nontumor tissues to identify transcripts that were altered in ESCC tissue. We performed molecular and functional studies of the transcripts identified and investigated the mechanisms of alteration. We identified protein tyrosine kinase 6 (PTK6) as a transcript that was significantly down-regulated in ESCC tissues and cell lines compared with nontumor tissues or immortalized normal esophageal cell lines. The promoter of the PTK6 gene was inactivated in ESCC tissues at least in part via hypermethylation and histone deacetylation. Knockdown of PTK6 in KYSE30 ESCC cells using small hairpin RNAs increased their ability to form foci, migrate, and invade extracellular matrix in culture and form tumors in nude mice. Overexpression of PTK6 in these cells reduced their proliferation in culture and tumor formation in mice. PTK6 reduced phosphorylation of Akt and glycogen synthase kinase (GSK)3β, leading to activation of β-catenin. PTK6 was identified as a transcript that is down-regulated in human ESCC tissues via epigenetic modification at the PTK6 locus. Its product appears to regulate cell proliferation by reducing phosphorylation of Akt and GSK3β, leading to activation of β-catenin. Reduced levels of PTK6 promote growth of xenograft tumors in mice; it might be developed as a marker of ESCC.

Ma S ，Bao JYJ ，Kwan PS ，Chan YP ，Tong CM ，Fu L ，Zhang N ，Tong AHY ，Qin YR ，Tsao SW ，Chan KW ，Lok S ，Guan XY ... -  《-》

Effect of Linc-POU3F3 on radiotherapy resistance and cancer stem cell markers of esophageal cancer cells.

Chen Y ，Tang J ，Li L ，Lu T ... -  《-》