基于高通量测序技术探究糖尿病足与健康人组织DNA中的5-羟甲基胞嘧啶变化  

To investigate the changes of 5-hydroxymethylcytosine in the DNA of patients with diabetic foot and healthy human via high-throughput sequencing technology

作　　者：马睿瑶 赵景会 储金林 铁璐 李念东[4] 李琳琳[1] MA Rui-yao;ZHAO Jing-hui;CHU Jin-lin;TIE Lu;LI Nian-dong;LI Lin-lin(School of Pharmacy,Xinjiang Medical University,Urumqi 830011,Xinjiang,China;Wound Healing Center,Peking University Third Hospital,Beijing 100191,China;School of Basic Medical Sciences,Peking University,Beijing 100191,China;School of Medical Engineering,Xinjiang Medical University,Urumqi 830011,Xinjiang,China)

机构地区：[1]新疆医科大学药学院,乌鲁木齐830011 [2]北京大学第三附属医院伤口治疗中心,北京100191 [3]北京大学基础医学院,北京100191 [4]新疆医科大学医学工程学院,乌鲁木齐830011

出　　处：《医学研究生学报》2022年第12期1268-1273,共6页Journal of Medical Postgraduates

基　　金：新疆维吾尔自治区高校科研计划(XJEDU2021I017)。

摘　　要：目的 5-羟甲基胞嘧啶(5hmC)作为表观遗传物质被广泛研究,但在糖尿病足的研究中较少。文中旨在研究糖尿病足患者(DFU)和健康志愿者组织基因组DNA中的5hmC的差异变化。方法 收集2021年1~12月就诊于北京大学第三附属医院确诊为DFU患者组织6例(DFU组)和Normal组织3例(对照组)。提取组织DNA,使用5hmC-Seal技术建立文库和Illumina高通量测序技术测序,筛选差异羟甲基化修饰基因(DhMGs)。采用STRING数据库构建蛋白互作网络,通过Cytoscape3.9.0软件绘制DhMGs的调控网络,并通过CytoHubba功能筛选出关键枢纽基因(Hub基因),采用DAVID数据库对Hub基因进行GO及KEGG富集分析,最后使用qRT-PCR和免疫组化进行验证。结果 共有166个上调基因和262个下调基因,其中包括NDUFB6、GALR1、GDAP1等已知的与糖尿病并发症相关的基因。DhMGs主要分布在内含子、基因间和启动子区域。通过CytoHubba分析可得到10个Hub DhMGs,分别是MAPK14、MAPK11、CDC42、MAP3K1、NCOA1、RAP1B、AGO4、RUNX1、HIF1A、PHC3。对10个Hub基因进行富集分析,在生物过程中涉及到VEGFR信号通路、Ras蛋白信号转导、肌细胞分化的正向调节以及与免疫相关的白细胞介素12分泌的正向调节等;在细胞成分中主要与细胞质、胞液等相关;在分子功能中主要有细分化调控、血管内皮生长因子受体信号通路、髓样细胞分化调控等功能。KEGG分析显示Hub基因主要富集于白细胞跨内皮细胞迁移、有丝分裂原活化蛋白激酶信号通路等。最后通过qRT-PCR和免疫组化实验得出MAPK11和CDC42在DFU组织中的表达水平均明显升高(P<0.05)。结论 糖尿病足和健康人组织DNA的5hmC修饰具有差异,MAPK11和CDC42可能成为DFU潜在的治疗靶点。Objective As an epigenetic material, 5-hydroxymethylcytosine(5-hmC) has been widely studied, but it is rarely studied in diabetic foot. This paper aims to study the changes of 5-hydroxymethylcytosine(5-hmC) in the DNA of diabetic foot patients(DFU) and healthy volunteers. Methods The tissues of 6 patients diagnosed with DFU in the Third Affiliated Hospital of Peking University from January to December 2021 and 3 healthy volunteers were collected. When the DNA was extracted, the library using 5-hmC-Seal technology and Illumina high-throughput sequencing technology was used for screening the differences of Hydroxymethylation-modifying genes(DhMGs). The STRING was used to build the protein interaction network, the regulatory network of DhMGs was drawn by Cytoscape 3.9.0, the Hub genes were selected by CytoHubba and conducted the Go/KEGG Enrichment Analysis by DAVID, and finally qRT-PCR and immunohistochemistry were verified. Results There were 166 up-regulated genes and 262 down-regulated genes, including the confirmed genes related to diabetic complications NDUFB6, GALR1, and GDAP1. DhMGs located in the intron, intergenic region, and promoter region. After CytoHubba analysis, there were ten Hub DhMGs of MAPK14, MAPK11, CDC42, MAP3K1, NCOA1, AGO4, RUNX1, HIF1A, and PHC3. The ten Hub genes went through Enrichment Analysis. The VEGFR signaling pathway, The Ras protein signal transduction, and positive regulation of myocyte differentiation and immune-related interleukin 12 were included in the biological process. The cytoplasm and cytosol were major components. The regulation of cell differentiation, the signaling of vascular endothelial growth factor(VEGF), and the regulation of myeloid cell differentiation were included in the molecular functions, KEGG analysis showed that hub genes mainly located in the pathways of leukocytes to endotheliocyte and mitogen-activated protein kinase, Finally, qRT-PCR and immunohistochemical experiments showed that MAPK11 and CDC42 were significantly increased in DFU tissues(P<0.05). Co

关 键 词：糖尿病足 5-羟甲基胞嘧啶 有丝分裂原活化蛋白激酶14 有丝分裂原活化蛋白激酶11 细胞分裂周期因子42 

分 类 号：R587.1[医药卫生—内分泌]