糖尿病足溃疡差异表达基因的筛选与功能分析及临床验证

王鹏; 陈昭宏; 蒋丽媛; 周小茜; 贾赤宇; 肖厚安

doi:10.3760/cma.j.cn501225-20220731-00328

糖尿病足溃疡差异表达基因的筛选与功能分析及临床验证

doi: 10.3760/cma.j.cn501225-20220731-00328

1.
西安市第九医院烧伤整形美容科，西安　　710054
2.
福建医科大学附属协和医院烧伤与创面修复科，福建省烧伤研究所，福建省烧伤医学中心，福建省烧创伤重点实验室，福州　　350001
3.
厦门大学附属翔安医院烧伤整形与创面修复科，厦门　　361102

基金项目:

陕西省重点研发计划 S2021-YF-YBSF-0936

西安市卫生健康委员会基金项目 2020yb21, 2022yb03, 2022yb04, 2022yb05

福建医科大学附属协和医院2020年省重点实验室开放课题 XHZDSYS202004

详细信息

通讯作者:
肖厚安，Email：xiaohouan@163.com

计量
- 文章访问数: 2611
- HTML全文浏览量: 41
- PDF下载量: 45
- 被引次数: 0
出版历程
- 收稿日期: 2022-07-31

Screening, functional analysis and clinical validation of differentially expressed genes in diabetic foot ulcers

1.
Department of Burns and Plastic and Cosmetic Surgery, Xi'an Ninth Hospital, Xi'an 710054, China
2.
Department of Burns and Wound Repair, Union Hospital, Fujian Medical University, Fujian Provincial Burn Research Institute, Fujian Burn Medical Center, Fujian Provincial Key Laboratory of Burn and Trauma, Fuzhou 350001, China
3.
Department of Burns and Plastic and Wound Repair Surgery, Xiang'an Hospital of Xiamen University, Xiamen 361102, China

Funds:

Key Research and Development Plan of Shaanxi Province of China S2021-YF-YBSF-0936

Xi'an Health Commission Fund Project 2020yb21, 2022yb03, 2022yb04, 2022yb05

2020 Provincial Key Laboratory Open Project of Fujian Medical University Affiliated Union Hospital XHZDSYS202004

More Information

Corresponding author: Xiao Hou'an, Email: xiaohouan@163.com

摘要

摘要: 目的筛选糖尿病足溃疡（DFU）的差异表达基因（DEG）并对其进行功能分析与临床验证，以期为慢性难愈性创面的表观遗传学治疗奠定理论基础。方法采用观察性研究方法。选取基因表达综合数据库（GEO）中的DFU患者基因表达谱数据集GSE80178，采用GEO2R工具分析筛选数据集中3个正常皮肤组织样本与6个DFU组织样本之间的DEG。对筛选出的DEG，采用R语言程序包中ClusterProfiler、org.Hs.eg.db、GOplot和ggplot2分别进行生物学过程、分子功能、细胞组分的基因本体论（GO）富集分析和京都基因与基因组百科全书（KEGG）富集分析；采用STRING数据库进行蛋白质-蛋白质相互作用（PPI）分析，筛选DEG中的关键基因，用Cytoscape 3.9.1软件中Cytohubba插件进行关键基因的GO富集分析。取厦门大学附属翔安医院2018年9月—2021年3月收治的15例DFU患者（男7例、女8例，年龄55~87岁）的DFU组织和15例急性创面患者（男6例、女9例，年龄8~52岁）术后弃用的正常皮肤组织，分别采用实时荧光定量反转录PCR法与免疫组织化学法检测富含脯氨酸的小重复蛋白1A（SPRR1A）和晚期角质化包膜蛋白3C（LCE3C）的mRNA与蛋白表达。对数据行独立样本t检验。结果与正常皮肤组织比较，从DFU患者DFU组织中筛选出492个差异表达显著的DEG（校正P<0.05或校正P<0.01），包括363个上调DEG和129个下调DEG。GO术语分析显示，DEG在皮肤发育、角质形成细胞（KC）分化、角质化、表皮发育、表皮细胞分化等方面显著富集（校正P值均<0.01）；KEGG通路分析显示，DEG在肿瘤相关微小RNA、Ras相关蛋白1信号通路和多能干细胞调控信号通路等方面显著富集（校正P值均<0.01）。PPI分析显示，内披蛋白、SPRR1A、SPRR1B、SPRR2B、SPRR2E、SPRR2F、LCE3C、LCE3E、角蛋白16（均为下调DEG）和丝聚蛋白（为上调DEG）为从DFU患者DFU组织中筛选出的DEG中的关键基因，其显著富集于角质化、KC分化、表皮细胞分化、皮肤发育、表皮发育、多肽交联等GO术语（校正P值均<0.01）。DFU患者DFU组织中SPRR1A和LCE3C的mRNA表达量分别为0.588±0.082与0.659±0.098、蛋白表达量分别为0.22±0.05与0.24±0.04，分别明显低于急性创面患者正常皮肤组织的1.069±0.025与1.053±0.044（t值分别为20.91、13.66，P值均<0.01）、0.38±0.04与0.45±0.05（t值分别为9.69、12.46，P值均<0.01）。结论相较于正常皮肤组织，DFU患者DFU组织中存在DEG谱，且DEG显著富集于KC分化及角蛋白功能方面；关键DEG与KC生物学功能相关，在DFU患者DFU组织中的低表达可能阻碍溃疡愈合。
- 糖尿病足 /
- 溃疡 /
- 计算生物学 /
- 创面修复 /
- 愈合机制 /
- 差异表达基因
Abstract: Objective To screen the differentially expressed genes (DEGs) in diabetic foot ulcers (DFUs), and to perform functional analysis and clinical validation of them, intending to lay a theoretical foundation for epigenetic therapy of chronic refractory wounds. Methods An observational study was conducted. The gene expression profile dataset GSE80178 of DFU patients in Gene Expression Omnibus (GEO) was selected, and the DEG between three normal skin tissue samples and six DFU tissue samples in the dataset was analyzed and screened using the GEO2R tool. For the screened DEG, ClusterProfiler, org.Hs.eg.db, GOplot, and ggplot2 in the R language packages were used for Gene Ontology (GO) enrichment analysis of biological processes, molecular functions, and cellular components, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, respectively. Protein-protein interaction (PPI) analysis was performed using STRING database to screen key genes in the DEG, and GO enrichment analysis of key genes was performed using Cytohubba plug-in in Cytoscape 3.9.1 software. DFU tissue and normal skin tissue discarded after surgery were collected respectively from 15 DFU patients (7 males and 8 females, aged 55-87 years) and 15 acute wound patients (6 males and 9 females, aged 8-52 years) who were admitted to Xiang'an Hospital of Xiamen University from September 2018 to March 2021. The mRNA and protein expressions of small proline-rich repeat protein 1A (SPRR1A) and late cornified envelope protein 3C (LCE3C) were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Data were statistically analyzed with independent sample t test. Results Compared with normal skin tissue, 492 statistically differentially expressed DEGs were screened from DFU tissue of DFU patients (corrected P<0.05 or corrected P<0.01), including 363 up-regulated DEGs and 129 down-regulated DEGs. GO terminology analysis showed that DEGs were significantly enriched in the aspects of skin development, keratinocyte (KC) differentiation, keratinization, epidermal development, and epidermal cell differentiation, etc. (corrected P values all <0.01). KEGG pathway analysis showed that DEGs were significantly enriched in the aspects of tumor-associated microRNA, Ras related protein 1 signaling pathway, and pluripotent stem cell regulatory signaling pathway, etc. (corrected P values all <0.01). PPI analysis showed that endophial protein, SPRR1A, SPRR1B, SPRR2B, SPRR2E, SPRR2F, LCE3C, LCE3E, keratin 16 (all down-regulated DEGs), and filoprotein (up-regulated DEG) were key genes of DEGs screened from DFU tissue of DFU patients, which were significantly enriched in GO terms of keratinization, KC differentiation, epidermal cell differentiation, skin development, epidermis development, and peptide cross-linking, etc. (corrected P values all <0.01). The mRNA expressions of SPRR1A and LCE3C in DFU tissue of DFU patients were 0.588±0.082 and 0.659±0.098, respectively, and the protein expressions were 0.22±0.05 and 0.24±0.04, respectively, which were significantly lower than 1.069±0.025 and 1.053±0.044 (with t values of 20.91 and 13.66, respectively, P values all <0.01) and 0.38±0.04 and 0.45±0.05 (with t values of 9.69 and 12.46, respectively, P values all <0.01) in normal skin tissue of acute wound patients. Conclusions Compared with normal skin tissue, there is DEG profile in DFU tissue of DFU patients, with DEGs being significantly enriched in the aspects of KC differentiation and keratin function. Key DEGs are related to the biological function of KC, and their low expressions in DFU tissue of DFU patients may impede ulcer healing.
- Diabetic foot /
- Ulcer /
- Computational biology /
- Wound repair /
- Healing mechanism /
- Differentially expressed genes
王鹏：实验操作、数据整理、论文撰写与经费支持；陈昭宏、贾赤宇：研究指导、论文修改、经费支持；蒋丽媛、周小茜：数据整理、数据分析；肖厚安：设计实验、对文章的知识性内容作批评性审阅、获取研究经费、行政与技术及材料支持

所有作者均声明不存在利益冲突

HTML全文

下载: 全尺寸图片幻灯片

1 DFU患者DFU组织相较于正常皮肤组织的DEG可视化火山图

注：FDR为错误发现率，差异倍数为糖尿病足溃疡（DFU）组织与正常皮肤组织基因表达量的比值；水平虚线向上为校正P<0.05的差异表达基因（DEG），蓝色散点为筛选出的下调DEG，红色散点为筛选出的上调DEG

下载: 全尺寸图片幻灯片

2 DFU患者DFU组织相较于正常皮肤组织的差异表达基因的基因本体论富集分析中富集程度排名前20者

注：DFU为糖尿病足溃疡，RAGE为晚期糖基化终末产物受体

下载: 全尺寸图片幻灯片

3 DFU患者DFU组织相较于正常皮肤组织的差异表达基因的京都基因与基因组百科全书富集分析中富集程度排名前20者

注：DFU为糖尿病足溃疡，JAK-STAT为酪氨酸蛋白激酶-信号转导及转录激活因子，Rap1为Ras相关蛋白1

下载: 全尺寸图片幻灯片

4 DFU患者DFU组织相较于正常皮肤组织的DEG的蛋白质-蛋白质相互作用分析中结合分数排名前100者

注：DFU为糖尿病足溃疡;红色为上调差异表达基因（DEG），绿色为下调DEG;四边形中DEG为最大集团中心性分数排名前10者，即关键基因；各图形中文字代表各基因名称

下载: 全尺寸图片幻灯片

5 免疫组织化学染色后观察DFU患者DFU组织与急性创面患者正常皮肤组织中SPRR1A和LCE3C的蛋白表达分布（均为棕黄色）二氨基联苯胺-苏木精×200。5A.正常皮肤组织可见细胞内较多SPRR1A蛋白分布；5B.DFU组织细胞内SPRR1A蛋白分布较图5A少；5C.正常皮肤组织可见细胞内较多LCE3C蛋白分布；5D.DFU组织细胞内LCE3C蛋白分布较图5C少

注：DFU为糖尿病足溃疡，SPRR1A为富含脯氨酸的小重复蛋白1A，LCE3C为晚期角质化包膜蛋白3C

下载: 全尺寸图片幻灯片

参考文献(38)

[1]	《多学科合作下糖尿病足防治专家共识(2020版)》编写组.多学科合作下糖尿病足防治专家共识(2020版)精华版(Ⅱ)[J].中华烧伤杂志,2020,36(9):767-785.DOI: 10.3760/cma.j.cn501120-20200217-00062.
[2]	ArmstrongDG,BoultonAJM,BusSA.Diabetic foot ulcers and their recurrence[J].N Engl J Med,2017,376(24):2367-2375.DOI: 10.1056/NEJMra1615439.
[3]	JiangY,WangX,XiaL,et al.A cohort study of diabetic patients and diabetic foot ulceration patients in China[J].Wound Repair Regen,2015,23(2):222-230.DOI: 10.1111/wrr.12263.
[4]	陈泓邑.生物医学大数据共享中的“信任”与“配享信任”[J].医学与哲学,2021,42(2):23-27.DOI: 10.12014/j.issn.1002-0772.2021.02.05.
[5]	姜林宏,张鹤达,钟山亮,等.基于GEO数据库分析circRNAs在胃癌组织中的表达[J].中国肿瘤外科杂志,2020,12(6):529-533.DOI: 10.3969/j.issn.1674-4136.2020.06.008.
[6]	张钧栋,杨波,刘霖,等.严重急性呼吸综合征冠状病毒感染肺损伤机制与干预药物的生物信息学分析[J].中华实验和临床病毒学杂志,2020,34(4):367-373.DOI: 10.3760/cma.j.cn112866-20200318-00074.
[7]	赵倩倩,尤红俊,肖丹,等.基于基因表达综合数据库芯片的类风湿关节炎生物标志物的筛选与生物信息学分析[J].西安交通大学学报(医学版),2020,41(4):544-552.DOI: 10.7652/jdyxb202004014.
[8]	王欣凯,王硕.探究生物信息学的研究进展[J].科技资讯,2020,18(14):228-229.DOI: 10.16661/j.cnki.1672-3791.2020.14.228.
[9]	闫小妮,田国祥,郭晓娟,等.GEO数据库架构、申请及数据提取方法与流程[J].中国循证心血管医学杂志,2019,11(2):134-137.DOI: 10.3969/j.issn.1674-4055.2019.02.02.
[10]	胡建雄,陈思齐,刘涛,等.R语言在公共卫生领域的应用:基本数据处理[J].华南预防医学,2020,46(2):183-184,188.DOI: 10.12183/j.scjpm.2020.0183.
[11]	CrosaraKTB,MoffaEB,XiaoY,et al.Merging in-silico and in vitro salivary protein complex partners using the STRING database: a tutorial[J].J Proteomics,2018,171:87-94.DOI: 10.1016/j.jprot.2017.08.002.
[12]	江珊,蒋勃,徐桂珍,等.使用Cytoscape对生物网络数据的建模和分析[J].农业网络信息,2017(6):32-37.DOI: 10.3969/j.issn.1672-6251.2017.06.009.
[13]	郭光华,朱峰,闵定宏,等.糖尿病足合并难愈性创面外科治疗全国专家共识(2020版)[J/CD].中华损伤与修复杂志:电子版,2020,15(4):256-263.DOI: 10.3877/cma.j.issn.1673-9450.2020.04.005.
[14]	申金付,蒋瑞妹,王卓群,等.2型糖尿病患者糖尿病足溃疡复发情况及影响因素[J].中华烧伤杂志,2020,36(10):947-952.DOI: 10.3760/cma.j.cn501120-20190726-00315.
[15]	DingX,TangQ,XuZ,et al.Challenges and innovations in treating chronic and acute wound infections: from basic science to clinical practice[J/OL].Burns Trauma,2022,10:tkac014[2022-07-31]. https://pubmed.ncbi.nlm.nih.gov/35611318/.DOI: 10.1093/burnst/tkac014.
[16]	RaghuramAC,YuRP,LoAY,et al.Role of stem cell therapies in treating chronic wounds: a systematic review[J].World J Stem Cells,2020,12(7):659-675.DOI: 10.4252/wjsc.v12.i7.659.
[17]	WangA,LvG,ChengX,et al.Guidelines on multidisciplinary approaches for the prevention and management of diabetic foot disease (2020 edition)[J/OL].Burns Trauma,2020,8:tkaa017[2022-07-31].https://pubmed.ncbi.nlm.nih.gov/32685563/.DOI: 10.1093/burnst/tkaa017.
[18]	马俊,吴霞,宫伟,等.生物信息学相关数据库在肿瘤研究中的应用[J].中国医药生物技术,2019,14(1):69-71.DOI: 10.3969/j.issn.1673-713X.2019.01.014.
[19]	林志琥,王君,梁尊鸿,等.干细胞治疗糖尿病足创面的研究进展[J].中华烧伤与创面修复杂志,2022,38(3):281-286.DOI: 10.3760/cma.j.cn501120-20210828-00292.
[20]	王雪欣,余鹏,张明谏,等.糖尿病足患者下肢动脉病变和伤口细菌学分析[J].中华烧伤杂志,2018,34(6):386-388.DOI: 10.3760/cma.j.issn.1009-2587.2018.06.014.
[21]	薛春利,胡志成,杨祖贤,等.异体脱细胞真皮基质治疗糖尿病足溃疡临床效果荟萃分析[J].中华烧伤杂志,2016,32(12):725-729.DOI: 10.3760/cma.j.issn.1009-2587.2016.12.005.
[22]	徐进,殷嫦嫦,宋伟,等.间充质干细胞治疗糖尿病足溃疡的Meta分析[J].中国糖尿病杂志,2019,27(6):417-423.DOI: 10.3969/j.issn.1006-6187.2019.06.004.
[23]	VerdiJ,ShirianS,SalehM,et al.Mesenchymal stem cells regenerate diabetic foot ulcers: a review article[J].World J Plast Surg,2022,11(1):12-22.DOI: 10.52547/wjps.11.1.12.
[24]	Álvaro-AfonsoFJ,Sanz-CorbalánI,Lázaro-MartínezJL,et al.Adipose-derived mesenchymal stem cells in the treatment of diabetic foot ulcers: a review of preclinical and clinical studies[J].Angiology,2020,71(9):853-863.DOI: 10.1177/0003319720939467.
[25]	任慧雯,王秋月.Epac-Rap1与糖尿病肾病[J].国际内分泌代谢杂志,2015,35(3):170-172.DOI: 10.3760/cma.j.issn.1673-4157.2015.03.007.
[26]	陆树良,谢挺,牛轶雯.创面难愈机制研究——糖尿病皮肤的“微环境污染”[J].中华烧伤杂志,2008,24(1):3-5.DOI: 10.3760/cma.j.issn.1009-2587.2008.01.002.
[27]	李校堃.坚持梦想不负韶华:生长因子与创面修复三十年自主创新之路[J].中华烧伤杂志,2020,36(3):161-165.DOI: 10.3760/cma.j.cn501120-20200305-00125.
[28]	董叫云,弓家弘,嵇晓芸,等.异体糖尿病大鼠来源脂肪干细胞移植治疗糖尿病大鼠创面的初步评价及其机制探讨[J].中华烧伤杂志,2019,35(9):645-654.DOI: 10.3760/cma.j.issn.1009-2587.2019.09.002.
[29]	KuritaM,AraokaT,HishidaT,et al.In vivo reprogramming of wound-resident cells generates skin epithelial tissue[J].Nature,2018,561(7722):243-247.DOI: 10.1038/s41586-018-0477-4.
[30]	RoshanA,MuraiK,FowlerJ,et al.Human keratinocytes have two interconvertible modes of proliferation[J].Nat Cell Biol,2016,18(2):145-156.DOI: 10.1038/ncb3282.
[31]	PiipponenM,LiD,LandénNX.The immune functions of keratinocytes in skin wound healing[J].Int J Mol Sci,2020,21(22):8790.DOI: 10.3390/ijms21228790.
[32]	RiouxG,RidhaZ,SimardM,et al.Transcriptome profiling analyses in psoriasis: a dynamic contribution of keratinocytes to the pathogenesis[J].Genes (Basel),2020,11(10):1155.DOI: 10.3390/genes11101155.
[33]	VermeijWP,BackendorfC.Skin cornification proteins provide global link between ROS detoxification and cell migration during wound healing[J].PLoS One,2010,5(8):e11957.DOI: 10.1371/journal.pone.0011957.
[34]	MizuguchiY,SpechtS,LunzJG3rd,et al.SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity[J].BMC Mol Biol,2012,13:20.DOI: 10.1186/1471-2199-13-20.
[35]	ZhangC,HuZ,LoneAG,et al.Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption[J].Elife,2022,11:e76729.DOI: 10.7554/eLife.76729.
[36]	NiehuesH,van der KriekenDA,EderveenTHA,et al.Antimicrobial late cornified envelope proteins: the psoriasis risk factor deletion of LCE3B/C genes affects microbiota composition[J].J Invest Dermatol,2022,142(7):1947-1955.e6.DOI: 10.1016/j.jid.2021.11.036.
[37]	ZhangX,YinM,ZhangLJ.Keratin 6, 16 and 17-critical barrier alarmin molecules in skin wounds and psoriasis[J].Cells,2019,8(8):807.DOI: 10.3390/cells8080807.
[38]	尹丽芬,柳志文,吴昊,等.丝聚蛋白在口腔黏膜下纤维性变中的表达[J].口腔疾病防治,2019,27(9):557-560.DOI: 10.12016/j.issn.2096-1456.2019.09.003.