NGALR Is Overexpressed And Regulated By Hypomethylation In Esophageal Squamous Cell Carcinoma

Neutrophil gelatinase-associated lipocalin (NGAL) is an important gene related with tumor, which was up-expressed in colon cancer, breast cancer, pancreatic cancer, lung cancer and esophageal cancer. Recently, a specific cell-surface receptor for 24p3/NGAL (24p3R/NGALR) was isolated from murine FL5.12 cells. It was reported that neutrophil gelatinase-associated lipocalin receptor (NGALR) mRNA level is reduced in chronic myelogenous leukemia blast isolates but up-regulated in esophageal squamous cell carcinoma (ESCC). The mechanism of NGALR regulation is unknown. Here we demonstrate the expression pattern of NGALR and examine the aberrant methylation of its gene in ESCC and esophageal carcinoma cell lines. Expression of NGALR protein was analyzed by immunohistochemistry in 59 paraffin sections of esophageal carcinoma tissue and the corresponding sections of normal esophageal epithelium tissue. Quantitation of immunohistochemical result revealed that the expression of NGALR in ESCC was significantly higher than that in normal esophageal epithelium (P<0.01). Expression of NGALR mRNA in ESCC tissues and cell lines was analyzed by RT-PCR. NGALR1 and NGALR2 mRNA expression was up-regulated in 14 cases and 15 cases respectively,both NGALR1 and NGALR2 had a T/N ratio >1.0 in 13 of 20 cases. NGALR mRNA level was abundant in three tumor cell lines but was undetectable in two other tumor cell lines and one normal immortalized esophageal cell line SHEE. The luciferase reporter gene expression vectors containing NGALR gene 5'flanking regulation region were constructed by subcloning the different length DNA fragments of NGALR gene 5'flanking regulation region into pGL3-Basic vector. In EC8712 cells, all of the NGALR promoter–luciferase constructs demonstrated good activities above the pGL3-Basic background. The NGALR promoter had high reporter gene activity in EC8712, in contrast to residual reporter gene activity in EC109. DNA methylation status was investigated by methylation-specific PCR and by bisulfite genomic sequencing in esophageal carcinoma cell lines and surgically resected samples. Methylated alleles were detected in those cell lines lacking NGALR expression. Methylation was not detected in the NGALR-expressing cell lines. Methylation was observed in 31 of 77 (40.3%) ESCC tumor samples and in 46 of 77 (59.7%) paired normal tissues, demonstrating statistically different methylation of NGALR between normal and tumor tissues (P<0.05). The demethylating agent (5-aza-2'-deoxycytidine) restored NGALR expression in three methylated lines having a hypermethylated and silenced NGALR promoter, although with varying efficiency. In conclusion, our results suggest that NGALR hypomethylation contributes to its expression in esophageal carcinomas and may play a role in the pathogenesis of esophageal carcinomas.