NPY甲基化在结直肠癌患者中的诊断意义

Diagnostic role of NPY methylation in patients with colorectal cancer

目的:越来越多的研究表明基因甲基化生物标志物在肿瘤病变中发挥重要作用。本研究旨在探讨神经肽Y (NPY)甲基化在结直肠癌(CRC)中的诊断意义。方法:利用生物信息学工具分析癌症基因组图谱中所有肿瘤m RNA、蛋白质以及甲基化表达,生存获益以及免疫细胞浸润状态。CRC中NPY甲基化进一步在肠癌组织、粪便样本及细胞系中得到验证。使用Sequenome Epi TYPER和定量PCR方法进行NPY甲基化检测。实时PCR和蛋白质印迹法检测细胞系中NPY表达。结果:生物信息学分析显示大部分癌中NPY甲基化水平升高(P<0.05)。此外,NPY转录表达与结肠癌CD4+T细胞、巨噬细胞、树突状细胞(P<0.05)明显相关。直肠癌中CD4+T细胞、中性粒细胞和NPY也获得了类似的结果(P<0.05)。我们研究发现NPY在CRC组织和粪便脱落细胞中高甲基化(P<0.05)。粪便NPY甲基化在肿瘤、肠息肉(包括腺瘤性、锯齿状和炎性息肉)及健康对照组人群中敏感性分别为82.5%vs 46.3%vs23.4%,特异性为76.6%。细胞系体内实验表明5-aza-2’-deoxycytidine下调NPY甲基化水平并恢复其m RNA水平(P<0.05)。结论:本研究表明NPY在CRC中高甲基化,粪便DNA中NPY甲基化在中国CRC患者中是一种潜在的无创诊断生物标志物。

Objectives: A growing number of studies have shown that methylation biomarkers play an important role in oncogenesis. This study aimed to explore the diagnostic role of neuropeptide Y(NPY) methylation in colorectal cancer(CRC). Methods: m RNA and protein expression, methylation, survival benefits, and immune cell infiltration were analyzed using bioinformatics tools across all tumors from The Cancer Genome Atlas. NPY methylation in CRC was further validated in CRC tissues, fecal samples, and cell lines. Analyses of NPY methylation were performed using Sequenome Epi TYPER and quantitative PCR. Retrieval of NPY expression in cell lines was tested using real-time PCR and western blotting. Results: Bioinformatic analysis showed that the methylation level of NPY increased in most carcinomas(P<0.05).Moreover, statistical correlations were observed between NPY transcriptional expression and CD4+ T cells, macrophages,and dendritic cells in colon cancer(P<0.05). Similar results were obtained for CD4+ T cells, neutrophils, and NPY in rectal cancer(P<0.05). Our results showed that NPY was hypermethylated in CRC tissues and fecal exfoliated cells(P<0.05).Fecal NPY methylation was observed in 82.5% sensitive for primary tumors, 46.3% for intestinal polyps(including adenomatous, serrated, and inflammatory polyps), and 23.4% of healthy controls. Overall, fecal NPY methylation was 76.6%specific. For cell lines, in vivo experiments demonstrated that 5-aza-2′-deoxycytidine downregulated the methylation of NPY and restored its m RNA level(P<0.05). Conclusions: This study indicates that NPY is hypermethylated in CRC, and that NPY methylation in fecal DNA is a potential noninvasive diagnostic biomarker for Chinese patients with CRC.