Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Clin Oncol. Feb 10, 2017; 8(1): 67-74
Published online Feb 10, 2017. doi: 10.5306/wjco.v8.i1.67
NDRG2 gene copy number is not altered in colorectal carcinoma
Anders Lorentzen, Cathy Mitchelmore
Anders Lorentzen, Cathy Mitchelmore, Eucaryotic Cell Biology, Department of Science and Environment, Roskilde University, 4000 Roskilde, Denmark
Author contributions: Lorentzen A conceived the idea of the study and performed the experimental analysis; Lorentzen A and Mitchelmore C were both involved in interpretation of data, drafting the article and revising it critically for important intellectual content.
Supported by The Danish Cancer Society, No. DP05117.
Institutional review board statement: Human genomic DNA was purchased from Biochain Inc., whose IRB is registered with the Office for Human Research Protections (OHRP) with the registration number IRB00008283.
Conflict-of-interest statement: There is no conflict of interest.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Cathy Mitchelmore, PhD, Associate Professor, Department of Science and Environment, Roskilde University, Universitetsvej 1, Postbox 260, 4000 Roskilde, Denmark. mitch@ruc.dk
Telephone: +45-46743201 Fax:+45-46743011
Received: June 15, 2016
Peer-review started: June 18, 2016
First decision: August 16, 2016
Revised: November 11, 2016
Accepted: December 27, 2016
Article in press: December 28, 2016
Published online: February 10, 2017
Processing time: 237 Days and 17.8 Hours
Abstract
AIM

To investigate if the down-regulation of N-myc Downstream Regulated Gene 2 (NDRG2) expression in colorectal carcinoma (CRC) is due to loss of the NDRG2 allele(s).

METHODS

The following were investigated in the human colorectal cancer cell lines DLD-1, LoVo and SW-480: NDRG2 mRNA expression levels using quantitative reverse transcription-polymerase chain reaction (qRT-PCR); interaction of the MYC gene-regulatory protein with the NDRG2 promoter using chromatin immunoprecipitation; and NDRG2 promoter methylation using bisulfite sequencing. Furthermore, we performed qPCR to analyse the copy numbers of NDRG2 and MYC genes in the above three cell lines, 8 normal colorectal tissue samples and 40 CRC tissue samples.

RESULTS

As expected, NDRG2 mRNA levels were low in the three colorectal cancer cell lines, compared to normal colon. Endogenous MYC protein interacted with the NDRG2 core promoter in all three cell lines. In addition, the NDRG2 promoter was heavily methylated in these cell lines, suggesting an epigenetic regulatory mechanism. Unaltered gene copy numbers of NDRG2 were observed in the three cell lines. In the colorectal tissues, one normal and three CRC samples showed partial or complete loss of one NDRG2 allele. In contrast, the MYC gene was amplified in one cell line and in more than 40% of the CRC cases.

CONCLUSION

Our study suggests that the reduction in NDRG2 expression observed in CRC is due to transcriptional repression by MYC and promoter methylation, and is not due to allelic loss.

Keywords: N-myc downstream-regulated gene 2; Colorectal carcinoma; MYC; Tumor suppressor; Allelic loss; Gene amplification; Copy number

Core tip: NDRG2 is a putative tumor suppressor gene whose expression is reduced in many cancer forms, including colorectal carcinoma (CRC). We set out therefore to investigate if down-regulation of NDRG2 expression was due to loss of one or both alleles and/or to other mechanisms. In our paper, we show that allelic loss of NDRG2 is a rare event in CRC. To our knowledge, this is the first study that has specifically investigated gene copy number of NDRG2 in CRC. Furthermore, our results suggest that MYC is amplified in more than 40% of CRC cases. MYC is known to repress transcription of NDRG2. Our results lead us to suggest that it is the transcriptional control of NDRG2 expression, including repression by MYC and epigenetic regulation, that results in decreased NDRG2 mRNA levels in CRC, rather than allelic loss of NDRG2.