摘要
AIM To investigate whether promoter methylation is re-sponsible for the silencing of formin 2 （ FMN2） in colorectal cancer （CRC） and to analyze the association between FMN2 methylation and CRC.METHODSWe frst identifed the expression levels and methylation levels of FMN2 in large-scale human CRC expression data-sets, including GEO and TCGA, and analyzed the relation-ship between the expression and methylation levels. Then, 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 inhibited DNA methylation 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.RESULTSA statistically significant downregulation of FMN2 ex-pression in large-scale human CRC expression datasets was found. Subsequent analysis showed that a high frequency of hypermethylation occurred in the FMN2 gene promoter in CRC tissues; operating characteristic curve analysis revealed that FMN2 gene methylation had a good capability for discriminating between CRC and nontumor tissue samples （AUC = 0.8432, P 〈 0.0001）. MethylTarget？ assays showed that CRC cells and tissues displayed higher methylation of these CpG regions than nontumor tissue samples. Correlation analysis showed a strong inverse correlation between methylation and FMN2 expression, and the inhibition of DNA methylation with 5-Aza significantly increased endogenous FMN2 expression. Analysis of the association between FMN2 methylation patterns and clinical indicators showed that FMN2 methylation was signifcantly associated with age, N stage, lymphovascular invasion, and pathologic tumor stage. Notably, 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 inva-sion, but the methylation level began to decrease with tumor progression. Additionally, FMN2 promoter hyper-methylation was more common in patients 〉 60 years old and in colon cancer tissue.CONCLUSIONFMN2 promoter hypermethylation 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.
AIM To investigate whether promoter methylation is responsible for the silencing of formin 2(FMN2) in colorectal cancer(CRC) and to analyze the association between FMN2 methylation and CRC. METHODS We first identified the expression levels and methylation levels of FMN2 in large-scale human CRC expression datasets, including GEO and TCGA, and analyzed the relationship between the expression and methylation levels. Then, 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 inhibited DNA methylation in CRC cells with 5-Aza-2'-deoxycytidine and assessed the expression of FMN2 by q RT-PCR. Last, the association between FMN2 methylation patterns and clinical indicators was analyzed.RESULTS A statistically significant downregulation of FMN2 expression in large-scale human CRC expression datasets was found. Subsequent analysis showed that a high frequency of hypermethylation occurred in the FMN2 gene promoter in CRC tissues; operating characteristic curve analysis revealed that FMN2 gene methylation had a good capability for discriminating between CRC and nontumor tissue samples(AUC = 0.8432, P < 0.0001). Methyl Target? assays showed that CRC cells and tissues displayed higher methylation of these CpG regions than nontumor tissue samples. Correlation analysis showed a strong inverse correlation between methylation and FMN2 expression, and the inhibition of DNA methylation with 5-Aza significantly increased endogenous FMN2 expression. Analysis of the association between FMN2 methylation patterns and clinical indicators showed that FMN2 methylation was significantly associated with age, N stage, lymphovascular invasion, and pathologic tumor stage. Notably, 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, but the methylation level began to decrease with tumor progression. Additionally, FMN2 promoter hypermethylation was more common in patients > 60 years old and in colon cancer tissue. CONCLUSION FMN2 promoter hypermethylation 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.
基金
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)
the New Xiangya Talent Projects of the Third Xiangya Hospital of Central South University,No.JY201508
