RNA-sequencing analysis of forearm skin in diabetic patients with or without foot ulcerations. RNA-sequencing analysis of forearm skin in diabetic patients with or without foot ulcerations

Diabetic foot ulcers (DFUs) and associated impaired healing, represent a major problem, that significantly impairs the quality of life of diabetic patients, leading to prolonged hospitalization and resulting in more than 70,000 lower extremity amputations per year in the USA alone. In the present study, we prospectively followed a large group of DFU patients for 12 weeks and aimed to identify systemic and local factors that are associated with DFU healing. We also studied healthy control subjects and diabetic patients without DFU and compared differences with the DFU patients. We first employed serum multiplex arrays to detect systemic cytokines, chemokines and growth factors, which correlate with DFU healing. In addition, we collected forearm biopsies for histology and bulk transcriptome analyses to establish whether DFU healing outcome was reflected at a non-ulcerative skin site. Bulk RNA-seq analysis revealed extracellular matrix (ECM) related genes up-regulated in Healers, including MMP2 as well as implication of IFNγ and IL13 as upstream regulators. According to transcriptome data analysis with a false discovery rate (FDR) 0.5, a total of 25 genes (3 up-regulated) were differentially expressed when comparing Non-Healers and Healers, 916 (530 up-regulated) in Healers compared to DM and 160 (89 up-regulated) in Non-Healers compared to DM. Genes of interest that were increased in Healers include inflammation associated molecules lymphoid chemokine ligand 19 (CCL19), complement component 6 (C6), lipoprotein lipase (LPL) and beta-defensin 124 (DEFB124), as well as extracellular matrix linked proteins pigment-epithelium derived factor (SERPINF1), tenascin X (TNXB), biglycan (BGN) and matrix metalloproteinase-2 (MMP2). For Non-Healers, up-regulated genes were cytochrome P450 family member (CYP1A1), prostaglandin transporter (SLCO2A1) and metabolism regulator G0/G1 switch gene 2 (G0S2). Overall design: mRNA profiles from skin isolated from the volar aspect of the forearm from 13 diabetic patients; 5 who healed their ulcers, 4 who didn't heal and 4 who had no ulcers.

糖尿病足溃疡（Diabetic foot ulcers, DFUs）及其伴随的愈合障碍是一项重大临床问题，可严重降低糖尿病患者的生活质量，导致住院周期延长，仅美国每年就有超过7万例下肢截肢事件由其引发。本研究前瞻性随访了一大队列DFU患者，随访时长为12周，旨在筛选与DFU愈合相关的全身与局部调控因素。同时纳入健康对照受试者与无DFU的糖尿病患者作为对照，与DFU患者组进行组间差异比较。本研究首先采用血清多重蛋白阵列技术，检测与DFU愈合相关的全身细胞因子、趋化因子及生长因子水平；此外采集前臂活检组织，开展组织学检测与批量转录组（bulk transcriptome）分析，以验证非溃疡皮肤部位是否可反映DFU的愈合结局。批量RNA测序（bulk RNA-seq）分析显示，愈合组受试者中存在与细胞外基质（extracellular matrix, ECM）相关的基因上调，包括基质金属蛋白酶2（matrix metalloproteinase-2, MMP2），并揭示干扰素γ（IFNγ）与白细胞介素13（IL13）作为上游调控因子的潜在作用。基于错误发现率（false discovery rate, FDR）为0.5的转录组数据分析，非愈合组与愈合组间共鉴定出25个差异表达基因（其中3个上调）；愈合组与无DFU的糖尿病（diabetes mellitus, DM）患者组相比，存在916个差异表达基因（其中530个上调）；非愈合组与DM患者组相比，存在160个差异表达基因（其中89个上调）。愈合组中上调的目标基因包括炎症相关分子：淋巴趋化因子配体19（CCL19）、补体成分6（C6）、脂蛋白脂肪酶（LPL）及β防御素124（DEFB124）；以及与细胞外基质相关的蛋白：色素上皮源性因子（SERPINF1）、肌腱蛋白X（TNXB）、饰胶蛋白（BGN）与基质金属蛋白酶2（MMP2）。非愈合组中上调的基因则包括细胞色素P450家族成员（CYP1A1）、前列腺素转运蛋白（SLCO2A1）及代谢调控基因G0/G1切换基因2（G0S2）。整体实验设计：采集13名糖尿病患者前臂掌侧皮肤的mRNA转录谱；其中5名患者溃疡完全愈合，4名患者溃疡未愈合，另有4名患者无溃疡病史。