Basic Study
Copyright ©The Author(s) 2018. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Nov 28, 2018; 24(44): 5013-5024
Published online Nov 28, 2018. doi: 10.3748/wjg.v24.i44.5013
Involvement of methylation-associated silencing of formin 2 in colorectal carcinogenesis
Dao-Jiang Li, Zhi-Cai Feng, Xiao-Rong Li, Gui Hu
Dao-Jiang Li, Xiao-Rong Li, Gui Hu, Department of Gastrointestinal Surgery, the Third Xiangya Hospital, Central South University, Changsha 410013, Hunan Province, China
Zhi-Cai Feng, Department of Burns and Plastic Surgery, the Third Xiangya Hospital, Central South University, Changsha 410013, Hunan Province, China
Author contributions: Li DJ, Li XR, and Hu G contributed to conceptualization and design of the study, analyzed the data, and coordinated the research; Li DJ and Feng ZC performed the majority of the experiments and analyzed the data; all authors drafted the article and made revisions related to the intellectual content of the manuscript, and approved the final version of the article to be published.
Supported by the National Nature Science Foundation of China, No. 81773130; the Fundamental Research Funds for the Central Universities of Central South University(the Key Projects of Postgraduate Independent Exploration and Innovation of Central South University, No. 2018zzts050); and the New Xiangya Talent Projects of the Third Xiangya Hospital of Central South University, No. JY201508.
Institutional review board statement: This study was reviewed and approved by the Ethics Committee of Central South University. Informed consent was obtained from all individual participants included in the study.
Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists.
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:
Corresponding author to: Gui Hu, PhD, Surgeon, Department of Gastrointestinal Surgery, the Third Xiangya Hospital, Central South University, No. 172, Tongzi Po Road, Changsha 410013, Hunan Province, China.
Telephone: +86-13908472002 Fax: +86-731-88618832
Received: August 20, 2018
Peer-review started: August 20, 2018
First decision: October 11, 2018
Revised: October 14, 2018
Accepted: November 7, 2018
Article in press: November 7, 2018
Published online: November 28, 2018
Research background

Colorectal cancer (CRC) is a critical contributor to cancer mortality and morbidity, and some critical genes and pathways were important in the initiation and progression of CRC. Recent studies have shown that formin 2 (FMN2) may be down-regulated in CRC. Whether FMN2 is abnormally expressed in CRC and what causes its abnormal expression are unclear. Revealing the role of FMN2 in CRC may provide potential therapeutic targets.

Research motivation

Epigenetic modifications of DNA, especially promoter hypermethylation, have critical roles in mediating some gene expression in the initiation and progression of CRC. Whether abnormal DNA methylation changes can occur in the promoter of the FMN2 gene in CRC and whether such changes can be responsible for the silencing of FMN2 are poorly known. So the main topic in this article is to try to solve this question that epigenetic silencing of FMN2 may be an important event in CRC. All can provide the basis and direction for future research.

Research objectives

This study focuses on whether FMN2 is underexpressed in CRC, and whether methylation changes occur in CpG islands located in the promoter region of FMN2. If changes occur, what are the characteristics of fragments and CpG sites in these CpG islands? What is the core fragment of the change? Does methylation change silence the expression of FMN2, and is there a correlation between methylation changes in FMN2 and clinical indicators of CRC? The answers to these questions can better explain the role of FMN2 in CRC.

Research methods

Large-scale human CRC expression datasets, including GEO and TCGA, were used to assess the expression levels and methylation levels of FMN2 in CRC. Then, the mRNA levels of FMN2 in our own clinical samples was analyzed by real-time quantitative polymerase chain reaction, and the methylation levels in four CpG regions adjacent to the FMN2 promoter were assessed by MethylTarget assays in CRC cells and in paired colorectal tumor samples and adjacent nontumor tissue samples. Furthermore, we performed demethylation treatment in CRC cells with 5-Aza-2’-deoxycytidine and assessed the expression of FMN2 by qRT-PCR. Last, the association between FMN2 methylation patterns and clinical indicators was analyzed.

Research results

FMN2 is underexpressed in CRC, and the most obvious low expression occurs in early colon cancer tissues. Subsequent analysis coming from large-scale human CRC datasets showed that FMN2 was one of the top 20 genes with an extremely high frequency of hypermethylation in CRC, methylation was the main somatic mutation, and FMN2 gene promoter hypermethylation occurred in 37.37% of CRC tissues. Our own experiments confirmed that CRC cells and tissues displayed higher methylation levels in CpG regions of FMN2 than nontumor tissue samples. Correlation analysis showed a strong inverse correlation between methylation and FMN2 expression. Treatment of CRC cells with demethylation reagent, 5-Aza-2’-deoxycytidine, can significantly increase endogenous FMN2 expression. The highest methylation of FMN2 occurred in tissues from cases of early-stage CRC, including cases with no regional lymph node metastasis (N0), cases in stages I and II, and cases with no lymphovascular invasion. Additionally, FMN2 promoter hypermethylation was more common in patients > 60 years and in colon cancer tissue.

Research conclusions

In this article we confirmed the low expression of FMN2 in CRC and first reported that a high frequency of hypermethylation occurred in the promoter of the FMN2 gene in CRC tissues and that hypermethylation was responsible for the silencing of FMN2. It is worth noting that we found that the low expression and hypermethylation of FMN2 occurred more prominently in early colon cancer tissues, which suggests that DNA hypermethylation leading to the low expression of the FMN2 gene may be an important early event in CRC, most likely playing a critical role in cancer initiation, and can serve as an ideal diagnostic biomarker in elderly patients with early-stage colon cancer.

Research perspectives

In the era of big data, the rational use of tumor databases such as TCGA, GEO, and COSMIC can provide us with valuable information. The emergence of some new technologies can provide more accurate data for our experiments. For example, MethylTarget assays developed by Genesky BioTech used in this article can be used very well for methylation analysis. Involvement of the methylation-associated silencing of FMN2 in colorectal carcinogenesis may be a valuable research direction. Future research can select more clinical specimens to explore whether it can be used as a good clinical diagnostic index, and can also develop demethylation treatment. The article on the relationship between FMN2 and CRC is rare, so further study investigating whether FMN2 participates in the process of colorectal carcinogenesis and its underlying mechanisms are very important.