目的 利用生物信息技术检索特发性肺纤维化(idiopathic pulmonary fibrosis, IPF)基因芯片数据,并结合体外实验,筛选与验证IPF相关的蛋白,为该疾病的药物开发提供潜在的作用靶点。方法 采用基因表据库(Gene Expression Omnibus,GEO) 检索“IPF”词条,下载GSE150910芯片数据,利用Bioconductor 软件包 DESeq2分析正常组和IPF组差异表达基因,对所获得差异基因进行基因本体论(gene ontology,GO)功能分析、日本京都基因与基因组百科全书(kyoto gene and genome encyclopedia,KEGG)通路分析、蛋白互作网络分析并作可视化处理。通过液质联用对差异基因对应的蛋白质进行定量;体外研究关键蛋白对α平滑肌肌动蛋白(α-SMA)分泌或形成的影响。结果 共筛选到69个与细胞外基质形成相关的差异表达基因,包括53个上调基因和16个下调基因;功能分析显示,差异基因主要涉及到细胞外基质的降解(degradation of the extracellular matrix)、胶原蛋白降解(collagen degradation)和胶原链三聚化(collagen chain trimerization)通路;蛋白互作网络分析和蛋白定量研究显示,与IPF相关的基因主要为金属蛋白酶(MMP-3, 9, ADAMTS14)和P4HA3等;金属蛋白酶抑制剂和P4HA3抑制剂能抑制肺纤维α-SMA分泌。结论 通过筛选差异表达基因, 明确相关基因和蛋白在IPF发生或发展过程中作用,为IPF新药研发提供新思路和方案。
Abstract
OBJECTIVE To retrieve idiopathic pulmonary fibrosis(IPF) gene chip data using bioinformatics technology and provide potential target proteins for development of drugs for IPF combined with in vitro assay. METHODS The Gene Expression Omnibus(GEO) was searched by the “idiopathic pulmonary fibrosis” entry. The GSE150910 microarray data was downloaded and the difference in the expressed genes between the normal group and the IPF group were analyzed using the Bioconductor package DESeq2. Differential genes were obtained for analysis of GO function, KEGG pathway, and protein interaction network and then underwent visualization treatment. The proteins corresponding to the differential genes were quantified by liquid chromatography-mass spectrometry. The effects of key proteins on α-SMA secretion or formation were studied in vitro. RESULTS A total of 69 differentially expressed genes were screened, including 53 up-regulated genes and 16 down-regulated genes; functional analysis showed that the main differential genes were related with extracellular matrix degradation, collagen degradation,and collagen chain trimerization pathways; protein interaction network analysis and protein quantification studies showed that the genes associated with IPF were mainly metalloproteinases(MMP-3, 9, ADAMTS14) and P4HA3, etc.; matrix metalloproteinase inhibitors and P4HA3 inhibitors could inhibit the secretion of α-SMA. CONCLUSION By screening differentially expressed genes. The roles of related genes and proteins can be clarified in the occurrence or development of IPF and provide new ideas and solutions for the development of new drugs for IPF.
关键词
特发性肺纤维化 /
生物信息技术 /
细胞外基质 /
纳升级液质联用
{{custom_keyword}} /
Key words
idiopathic pulmonary fibrosis(IPF) /
bioinformatics /
extracellular matrix(ECM) /
NANO-LC-MS/MS
{{custom_keyword}} /
中图分类号：
R966
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] GLASS D S, GROSSFELD D, RENNA H A, et al. Idiopathic pulmonary fibrosis: Current and future treatment[J]. Clin Respir J, 2022, 16(2):84-96.
[2] COTTIN V, SPAGNOLO P, BONNIAUD P, et al. Mortality and respiratory-related hospitalizations in idiopathic pulmonary fibrosis not treated with antifibrotics[J]. Front Med(Lausanne), 2021, 8:802989.doi: 10.3389/fmed.2021.802989.
[3] RICHELDI L, COLLARD H R, JONES M G. Idiopathic pulmonary fibrosis[J]. Lancet, 2017,389(10082):1941-1952.
[4] FINNERTY J P, PONNUSWAMY A, DUTTA P, et al. Efficacy of antifibrotic drugs, nintedanib and pirfenidone, in treatment of progressive pulmonary fibrosis in both idiopathic pulmonary fibrosis(IPF) and non-IPF: a systematic review and meta-analysis[J]. BMC Pulm Med, 2021, 21(1):411-422.
[5] LACEDOIA D, CORREALE M, TRICARICO L, et al. Survival of patients with idiopathic pulmonary fibrosis and pulmonary hypertension under therapy with nintedanib or pirfenidone[J]. Intern Emerg Med, 2022, 17(3):815-822.
[6] WU W, QIU L, WU J, et al. Efficacy and safety of pirfenidone in the treatment of idiopathic pulmonary fibrosis patients: a systematic review and meta-analysis of randomised controlled trials[J]. BMJ Open, 2021, 11(12):e050004.Doi: 10.1136/bmjopen-2021-050004.
[7] RASMUSSEN H S, MCCANN P P. Matrix metalloproteinase inhibition as a novel anticancer strategy: a review with special focus on batimastat and marimastat[J]. Pharmacol Ther, 1997, 75(1):69-75.
[8] SCOTT K A, WOOD E, KARRAN E H. A matrix metalloproteinase inhibitor which prevents fibroblast-mediated collagen lattice contraction[J]. FEBS Lett, 1990, 441(1):137-140.
[9] LUO Y, XU W, CHEN H, et al. A novel profibrotic mechanism mediated by TGFβ-stimulated collagen prolyl hydroxylase expression in fibrotic lung mesenchymal cells[J]. J Pathol, 2015, 236(3): 384-394.
[10] UGAI K, MATSUDA S, MIKAMI H, et al. Inhibition of the SET8 pathway ameliorates lung fibrosis even through fibroblast dedifferentiation[J]. Front Mol Biosci, 2020, 7:192.Doi: 10.3389/fmolb.2020.00192.
[11] TIAN C M, FU L P, XIA D Z, et al. Exploration into the mechanism of effective components and potential targets of astragali radix in treatment of diabetic nephropathy based on network pharmacology and cell biology verification[J]. Chin Pharm J(中国药学杂志), 2022, 57(1): 52-61.
[12] STAAB-WEIJINTZ C A. Fighting the fiber: targeting collagen in lung fibrosis[J]. Am J Respir Cell Mol Biol,2022,66(4):363-381.
[13] CRAIG V J, ZHANG L, HAGOOD J S, et al. Matrix metalloproteinases as therapeutic targets for idiopathic pulmonary fibrosis[J]. Am J Respir Cell Mol Biol, 2015, 53(5):585-600.
[14] CAI Y, ZHU L, ZHANG F, et al. Noninvasive monitoring of pulmonary fibrosis by targeting matrix metalloproteinases(MMPs)[J]. Mol Pharm, 2013, 10(6):2237-2247.
[15] MCKEOWN S, RICHTER A G, O′KANE C, et al. MMP expression and abnormal lung permeability are important determinants of outcome in IPF[J]. Eur Respir J, 2009, 33(1):77-84.
[16] ESPINDOLA M S, HABIEL D M, COELHO A L, et al. Differential responses to targeting matrix metalloproteinase 9 in idiopathic pulmonary fibrosis[J]. Am J Respir Crit Care Med, 2021, 203(4):458-470.
[17] BETSUYAKU T, FYKUDA Y, PARKS W C, et al. Gelatinase B is required for alveolar bronchiolization after intratracheal bleomycin[J]. Am J Pathol, 2000, 157(2):525-535.
[18] MAEDA S, DEAN D, GOMEZ R,et al. The first stage of transforming growth factor β1 activation is release of the large latent complex from the extracellular matrix of growth plate chondrocytes by matrix vesicle stromelysin-1(MMP-3)[J]. Calcif Tissue Int, 2002, 70(1):54-65.
[19] YAMASHITA C M, DOLGONOS L, ZEMANS R L, et al. Matrix metalloproteinase 3 is a mediator of pulmonary fibrosis[J]. Am J Pathol, 2011, 179(4):1733-1745.
[20] BONNANS C, CHOU J, WERB Z. Remodelling the extracellular matrix in development and disease[J]. Nat Rev Mol Cell Biol, 2014, 15(12):786-801.
[21] APTE S S. A disintegrin-like and metalloprotease(reprolysin-type) with thrombospondin type 1 motif(ADAMTS) superfamily: functions and mechanisms[J]. J Biol Chem, 2009, 284(46):31493-31497.
[22] CEYLAN ONUESAL E D, ANGELIDIS I, SCHILLER H B, et al. Collagen biosynthesis, processing, and maturation in lung ageing[J]. Front Med, 2021, 8: 593874.Doi: 10.3389/fmed.2021.593874.
[23] HOLM NIELSEN S, WILLUMSEN N, LEEMING D J, et al. Serological assessment of activated fibroblasts by alpha-smooth muscle actin(α-SMA): A noninvasive biomarker of activated fibroblasts in lung disorders[J]. Transl Oncol, 2019, 12(2):368-374.
[24] AKAMASTSU T, ARAI Y, KOSUGI I, et al. Direct isolation of myofibroblasts and fibroblasts from bleomycin-injured lungs reveals their functional similarities and differences[J]. Fibrogenes Tissue Repair, 2013, 6(1):1-17.
[25] LUO Y, XU W, CHEN H, et al. A novel profibrotic mechanism mediated by TGF β-stimulated collagen prolyl hydroxylase expression in fibrotic lung mesenchymal cells[J]. J Pathol, 2015, 236(3):384-394.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
药物高通量筛选技术国家地方联合工程研究中心开放基金项目资助(M20202006;K20202001)
{{custom_fund}}
PDF(4514 KB)
