Zhang Cheng, Li Yong-Zhi, Dai Dong-Qiu
Department of Gastrointestinal Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, China.
Front Mol Biosci. 2021 Sep 10;8:645470. doi: 10.3389/fmolb.2021.645470. eCollection 2021.
The prognosis of gastric cancer (GC) patients is poor. The effect of aberrant DNA methylation on FOXF2 expression and the prognostic role of FOXF2 methylation in GC have not yet been identified. The RNA-Seq and gene methylation HM450 profile data were used for analyzing FOXF2 expression in GC and its association with methylation level. Bisulfite sequencing PCR (BSP) was performed to measure the methylation level of the FOXF2 promoter region in GC cell lines and normal GES-1 cells. The cells were treated with the demethylation reagent 5-Aza-dC, and the mRNA and protein expression levels of FOXF2 were then measured by qRT-PCR and western blot assays. The risk score system from SurvivalMeth was calculated by integrating the methylation level of the cg locus and the corresponding Cox regression coefficient. FOXF2 was significantly downregulated in GC cells and tissues. On the basis of RNA-Seq and Illumina methylation 450 data, FOXF2 expression was significantly negatively correlated with the FOXF2 methylation level (Pearson's R = -0.42, < 2.2e). The FOXF2 methylation level in the high FOXF2 expression group was lower than that in the low FOXF2 expression group. The BSP assay indicated that the methylation level of the FOXF2 promoter region in GC cell lines was higher than that in GES-1 cells. The qRT-PCR and western blot assay showed that FOXF2 mRNA and protein levels were increased in GC cells following treatment with 5-Aza-Dc. The methylation risk score model indicated that patients in the high risk group had poorer survival probability than those in the low risk group (HR = 1.84 (1.11-3.07) and = 0.0068). FOXF2 also had a close transcriptional regulation network with four miRNAs and their corresponding target genes. Functional enrichment analysis of the target genes revealed that these genes were significantly related to several important signaling pathways. FOXF2 was downregulated due to aberrant DNA methylation in GC, and the degree of methylation in the promoter region of FOXF2 was related to the prognosis of patients. The FOXF2/miRNAs/target genes axis may play a vital biological regulation role in GC.
胃癌（GC）患者的预后较差。异常DNA甲基化对FOXF2表达的影响以及FOXF2甲基化在胃癌中的预后作用尚未明确。利用RNA测序和基因甲基化HM450谱数据来分析FOXF2在胃癌中的表达及其与甲基化水平的关联。采用亚硫酸氢盐测序PCR（BSP）法检测胃癌细胞系和正常GES-1细胞中FOXF2启动子区域的甲基化水平。用去甲基化试剂5-氮杂-2'-脱氧胞苷（5-Aza-dC）处理细胞，然后通过qRT-PCR和蛋白质免疫印迹法检测FOXF2的mRNA和蛋白质表达水平。通过整合cg位点的甲基化水平和相应的Cox回归系数，计算来自SurvivalMeth的风险评分系统。FOXF2在胃癌细胞和组织中显著下调。基于RNA测序和Illumina甲基化450数据，FOXF2表达与FOXF2甲基化水平呈显著负相关（Pearson相关系数R = -0.42，P < 2.2e）。高FOXF2表达组的FOXF2甲基化水平低于低FOXF2表达组。BSP分析表明，胃癌细胞系中FOXF2启动子区域的甲基化水平高于GES-1细胞。qRT-PCR和蛋白质免疫印迹分析表明，用5-Aza-Dc处理后，胃癌细胞中FOXF2的mRNA和蛋白质水平升高。甲基化风险评分模型表明，高风险组患者的生存概率低于低风险组（风险比HR = 1.84（1.11 - 3.07），P = 0.0068）。FOXF2还与四个微小RNA及其相应的靶基因有密切的转录调控网络。对靶基因的功能富集分析表明，这些基因与几个重要的信号通路显著相关。在胃癌中，FOXF2因异常DNA甲基化而下调，FOXF2启动子区域的甲基化程度与患者的预后相关。FOXF2/微小RNA/靶基因轴可能在胃癌中发挥重要的生物学调节作用。
