NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer.
BACKGROUND
Nasopharyngeal carcinoma associated gene 6 (NGX6) is down-regulated in most colon cancer cell lines and tumor tissues when compared with their normal tissue samples. As a novel suppress tumor gene, it could inhibit colon cancer cell growth and cell cycle progression. However, little is known about the transcriptional mechanisms controlling NGX6 gene expression. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumorigenesis of colorectal carcinoma (CRC). In this study, we explored the role of DNA methylation in regulation of NGX6 transcription.


METHODS
In the present study, we cloned the NGX6 promoter with characteristics of a CpG island by luciferase reporter assay. Then, the CpG methylation status around the NGX6 promoter region in colon cancer cell lines and colorectal tumor tissues was examined by methylation-specific PCR and bisulfite DNA sequencing. Finally, 5-Aza-2'-deoxycytidine (5-Aza-dC) treatment was used to confirm the correlation between NGX6 promoter methylation and its gene inactivation.


RESULTS
The sequence spanning positions -157 to +276 was identified as the NGX6 promoter, in which no canonical TATA boxes were found, while two CAAT boxes and GC boxes were discovered. Methylation status was observed more frequently in 40 colorectal cancer samples than in 40 adjacent normal mucosa samples (18/40 versus 7/40; P < 0.05). An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the NGX6 promoter was associated with colorectal cancer patients age (P < 0.05). Moreover, a trend was shown toward metastasis status and primary site in colorectal carcinomas with NGX6 promoter methylation (p = 0.056 and P = 0.067, respectively). In addition, 5-Aza-dC could induce NGX6 mRNA expression and NGX6 promoter demethylation in HT-29 cells.


CONCLUSIONS
Down-regulation of NGX6 gene is related to the promoter methylation. DNA methylation of NGX6 promoter might be a potential molecular marker for diagnosis or prognosis, or serve as a therapeutic target.
Liu
Minji
M
Department of Gastroenterology, The Third Affiliated Hospital of Xiang Ya School of Medicine, Central South University, Changsha 410013, Hunan, PR China.
Peng
Ya
Y
Wang
Xiaoyan
X
Guo
Qin
Q
Shen
Shourong
S
Li
Guiyuan
G
D000367:Age Factors; D000368:Aged; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D016009:Chi-Square Distribution; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D018899:CpG Islands; D019175:DNA Methylation / Q000187:drug effects; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000077209:Decitabine; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D019073:HT29 Cells; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D009367:Neoplasm Staging; D011401:Promoter Regions, Genetic / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
D014408:Biomarkers, Tumor; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; C477325:TMEM8B protein, human; D025521:Tumor Suppressor Proteins; D000077209:Decitabine; D015254:DNA Modification Methylases; D001374:Azacitidine