Mining of differentially expressed genes of venous leg ulcers and screening of target genes_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LIU Guoquan ¹ ,  WANG Yannan ² , LIU Tianjiao ¹

1. School of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Ji’nan 250014, P. R. China;
2. Department of Peripheral Vascular Disease, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Ji’nan 250014, P. R. China;

Corresponding?author：

WANG Yannan, Email: hhl003@163.com

Keywords：

venous leg ulcer; bioinformatics; differentially expressed gene; data mining; molecular mechanism

DOI：

10.7507/1007-9424.202006097

Video：

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Objective To explore the differentially expressed genes (DEGs) in venous leg ulcer (VLU) by bioinformatics, and further explore the molecular mechanism of the disease, predict early diagnostic markers and treatment targets.Methods The expression profiles of VLU were downloaded from the gene expression omnibus (GEO) database, the DEGs of VLU and inflammatory phase of normal skin healing were identified by R software and used to perform gene ontology (GO) and KEGG pathway enrichment analysis, obtaining the key genes of the pathway. We analyzed the proteins of protein interaction (PPI) network by STRING database and Cytoscape 3.2.1 software to obtain hub genes.Results A total of 409 DEGs were obtained, including 173 upregulted genes and 236 downregulted genes. The GO analysis showed that the upregulated DEGs mainly distributed in collagen-containing extracellular matrix (ECM), cornified envelope and collagen trimer, involved in biological processes such as skin development, keratinocyte differentiation and cornification, which mediated molecular functions such as ECM structural constituent, ECM structural constituent conferring tensile strength and integrin binding. The downregulated DEGs mainly distributed in tertiary granule, secretory granule membrane and tertiary granule membrane cornification, involved in biological processes such as response to chemokine, leukocyte migration and neutrophil chemotaxis, which mediated molecular functions such as chemokine activity, chemokine receptor binding and cytokine activity. KEGG pathway enrichment analysis results showed that the upregulated DEGs were mainly enriched in ECM-receptor interaction and protein digestion and absorption pathways, collagen type Ⅰ alpha1 chain (COL1A1), collagen type Ⅰ alpha2 chain (COL1A2), and collagen type Ⅵ alpha 6 chain (COL6A6) were the key genes of pathway; the downregulated DEGs were mainly enriched in Staphylococcus aureus infection, Toll-like receptor signaling pathway and leukocyte transendothelial migration pathways, interleukin (IL)-1β, C-X-C motif chemokine ligand 8 (CXCL8), IL-10, matrix metalloproteinase (MMP)1, and MMP9 were the key genes of pathway. The hub core genes of the PPI network were formyl peptide receptor (FPR)1, FPR2, IL-1β, IL-10, and CXCL8.Conclusions The results of this study indicate that the genes and signaling pathways involved in COL1A1, COL1A2, COL6A6, IL-1β, CXCL8, IL-10, MMP1, and MMP9 affect the healing of VLU. FPR1, FPR2, IL-1β, IL-10, and CXCL8 can be used as potential therapeutic targets.

Citation： LIU Guoquan, WANG Yannan, LIU Tianjiao. Mining of differentially expressed genes of venous leg ulcers and screening of target genes. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2021, 28(4): 445-451. doi: 10.7507/1007-9424.202006097 Copy

1.	Lal, BK. Venous ulcers of the lower extremity: definition, epidemiology, economic and social burden. Semin Vasc Surg, 2015, 28(1): 3-5.
2.	中華醫學會外科學分會血管外科學組, 中國醫師協會血管外科醫師分會, 中國醫療保健國際交流促進會血管外科分會, 等. 中國慢性靜脈疾病診斷與治療指南. 中華醫學雜志, 2019, 99(39): 3047-3061.
3.	Stone RC, Stojadinovic O, Rosa AM, et al. A bioengineered living cell construct activates an acute wound healing response in venous leg ulcers. Sci Transl Med, 2017, 9(371): eaaf8611.
4.	Guest JF, Fuller GW, Vowden P. Venous leg ulcer management in clinical practice in the UK: costs and outcomes. Int Wound J, 2018, 15(1): 29-37.
5.	Vivas A, Lev-Tov H, Kirsner RS. Venous leg ulcers. Ann Intern Med, 2016, 165(3): ITC17-ITC32.
6.	Barrett T, Wilhite SE, Ledoux P, et al. NCBI GEO: archive for functional genomics data setsupdate. Nucleic Acids Res, 2013, 41(Database issue): D991-D995.
7.	Gautier L, Cope L, Bolstad BM, et al. Affy-analysis of affymetrix GeneChip data at the probe level. Bioinformatics, 2004, 20(3): 307-315.
8.	Ritchie ME, Phipson B, Wu D, et al. Limma powers differential expression analyses for RNA sequencing and microarray studies. Nucleic Acids Res, 2015, 43(7): e477.
9.	Ginestet C. Ggplot2: elegant graphics for data analysis. J R Stat Soc Stat, 2011, 174(1): 245.
10.	Yu G, Wang LG, Han Y, et al. ClusterProfiler: an R package for comparing biological themes among gene clusters. Omics, 2012, 16(5): 284-287.
11.	Harris MA, Clark J, Ireland A, et al. The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res, 2004, 32(Database issue): D258-D261.
12.	Kanehisa M. The KEGG database. Novartis Found Symp, 2002, 247: 91-101, 101-103, 119-128, 244-252.
13.	Szklarczyk D, Gable AL, Lyon D, et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res, 2019, 47(D1): D607-D613.
14.	Smoot ME, Ono K, Ruscheinski J, et al. Cytoscape 2. 8: new features for data integration and network visualization. Bioinformatics, 2011, 27(3): 431-432.
15.	Chin CH, Chen SH, Wu HH, et al. CytoHubba: identifying hub objects and sub-networks from complex interactome. BMC Systems Biol, 2014, 8 Suppl 4(Suppl 4): S11.
16.	Sood A, Granick MS, Tomaselli NL. Wound dressings and comparative effectiveness data. Adv Wound Care (New Rochelle), 2014, 3(8): 511-529.
17.	Liu YC, Margolis DJ, Isseroff RR. Does inflammation have a role in the pathogenesis of venous ulcers: A critical review of the evidence. J Invest Dermatol, 2011, 131(4): 818-827.
18.	Blumberg SN, Maggi J, Melamed J, et al. A histopathologic basis for surgical debridement to promote healing of venous ulcers. J Am Coll Surg, 2012, 215(6): 751-757.
19.	Filkor K, Németh T, Nagy I, et al. The expression of inflammatory cytokines, TAM tyrosine kinase receptors and their ligands is upregulated in venous leg ulcer patients: a novel insight into chronic wound immunity. Int Wound J, 2016, 13(4): 554-562.
20.	Crawford JM, Lal BK, Durán, WN, et al. Pathophysiology of venous ulceration. J Vasc Surg Venous Lymphati Disord, 2017, 5(4): 596-605.
21.	Chin JS, Madden L, Chew SY, et al. Drug therapies and delivery mechanisms to treat perturbed skin wound healing. Adv Drug Deliv Rev, 2019, 149-150: 2-18.
22.	Tian Y, Li H, Gao Y, et al. Quantitative proteomic characterization of lung tissue in idiopathic pulmonary fibrosis. Clin Proteomics, 2019, 16: 6.
23.	Simka M, Rybak Z. Hypothetical molecular mechanisms by which local iron overload facilitates the development of venous leg ulcers and multiple sclerosis lesions. Med Hypotheses, 2008, 71(2): 293-297.
24.	Okabe Y, Medzhitov R. Tissue biology perspective on macrophages. Nat Immunol, 2015, 17(1): 9-17.
25.	Cairo G, Recalcati S, Mantovani A, et al. Iron trafficking and metabolism in macrophages: contribution to the polarized phenotype. Trends Immunol, 2011, 32(6): 241-247.
26.	Wilkinson HN, Roberts ER, Stafford AR, et al. Tissue iron promotes wound repair via M2 macrophage polarisation and the chemokines CCL17 and CCL22. Am J Pathol, 2019, 189(11): 2196-2208.
27.	Moghaddam AS, Mohammadian S, Vazini H, et al. Macrophage plasticity, polarization and function in health and disease. J Cell Physiol, 2018, 233(9): 6425-6440.
28.	Yang HL, Tsai YC, Korivi M, et al. Lucidone promotes the cutaneous wound healing process via activation of the PI3K/AKT, Wnt/β-catenin and NF-κB signaling pathways. Biochim Biophys Acta Mol Cell Res, 2017, 1864(1): 151-168.
29.	Weinheimer-Haus EM, Mirza RE, Koh TJ. Nod-like receptor protein-3 inflammasome plays an important role during early stages of wound healing. PLoS One, 2015, 10(3): e0119106.
30.	Pukstad BS, Ryan L, Flo T H, et al. Non-healing is associated with persistent stimulation of the innate immune response in chronic venous leg ulcers. J Dermatol Sci, 2010, 59(2): 115-122.
31.	Chen L, Guo S, Ranzer MJ, et al. Toll-like receptor 4 has an essential role in early skin wound healing. J Invest Dermatol, 2013, 133(1): 258-267.
32.	Weyrich AS, Zimmerman GA. Platelets: signaling cells in the immune continuum. Trends Immunol, 2004, 25(9): 289-495.
33.	Chiraz A, Fatma G, Dhafer L. Role of human macrophage polarization in inflammation during infectious diseases. Int J Mol Sci, 2018, 19(6): 1801.
34.	Naik S, Larsen SB, Gomez NC, et al. Inflammatory memory sensitizes skin epithelial stem cells to tissue damage. Nature, 2017, 550(7677): 475-480.
35.	Romagnani P, Lasagni L, Annunziato F, et al. CXC chemokines: the regulatory link between inflammation and angiogenesis. Trends Immunol, 2004, 25(4): 201-209.
36.	Rennekampff HO, Hansbrough JF, Kiessig V, et al. Bioactive interleukin-8 is expressed in wounds and enhances wound healing. J Surg Res, 2000, 93(1): 41-54.
37.	Schultz G, Wysocki A. Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen, 2009, 17(2): 153-162.
38.	Krishna NA, Pennington HM, Coppola CD, et al. Connecting local and global sensitivities in a mathematical model for wound healing. Bull Math Biol, 2015, 77(12): 2294-2324.
39.	Tolg C, Liu M, Cousteils K, et al. Cell-specific expression of the transcriptional regulator RHAMM provides a timing mechanism that controls appropriate wound re-epithelialization. J Biol Chem, 2020, 295(16): 5427-5448.
40.	Raffetto JD. Pathophysiology of wound healing and alterations in venous leg ulcers-review. Phlebology, 2016, 31(1 Suppl): 56-62.
41.	Liew PX. The neutrophil's role during health and disease. Physiol Rev, 2019, 99(2): 1223-1248.
42.	Mirastschijski U, Haaksma CJ, Tomasek JJ, et al. Matrix metalloproteinase inhibitor GM 6001 attenuates keratinocyte migration, contraction and myofibroblast formation in skin wounds. Exp Cell Res, 2004, 299(2): 265-275.
43.	Prat C, Haas PJ, Bestebroer J, et al. A homolog of formyl peptide receptor-like 1 (FPRL1) inhibitor from Staphylococcus aureus (FPRL1 inhibitory protein) that inhibits FPRL1 and FPR. J Immunol, 2009, 183(10): 6569-6578.

1. Lal, BK. Venous ulcers of the lower extremity: definition, epidemiology, economic and social burden. Semin Vasc Surg, 2015, 28(1): 3-5.
2. 中華醫學會外科學分會血管外科學組, 中國醫師協會血管外科醫師分會, 中國醫療保健國際交流促進會血管外科分會, 等. 中國慢性靜脈疾病診斷與治療指南. 中華醫學雜志, 2019, 99(39): 3047-3061.
3. Stone RC, Stojadinovic O, Rosa AM, et al. A bioengineered living cell construct activates an acute wound healing response in venous leg ulcers. Sci Transl Med, 2017, 9(371): eaaf8611.
4. Guest JF, Fuller GW, Vowden P. Venous leg ulcer management in clinical practice in the UK: costs and outcomes. Int Wound J, 2018, 15(1): 29-37.
5. Vivas A, Lev-Tov H, Kirsner RS. Venous leg ulcers. Ann Intern Med, 2016, 165(3): ITC17-ITC32.
6. Barrett T, Wilhite SE, Ledoux P, et al. NCBI GEO: archive for functional genomics data setsupdate. Nucleic Acids Res, 2013, 41(Database issue): D991-D995.
7. Gautier L, Cope L, Bolstad BM, et al. Affy-analysis of affymetrix GeneChip data at the probe level. Bioinformatics, 2004, 20(3): 307-315.
8. Ritchie ME, Phipson B, Wu D, et al. Limma powers differential expression analyses for RNA sequencing and microarray studies. Nucleic Acids Res, 2015, 43(7): e477.
9. Ginestet C. Ggplot2: elegant graphics for data analysis. J R Stat Soc Stat, 2011, 174(1): 245.
10. Yu G, Wang LG, Han Y, et al. ClusterProfiler: an R package for comparing biological themes among gene clusters. Omics, 2012, 16(5): 284-287.
11. Harris MA, Clark J, Ireland A, et al. The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res, 2004, 32(Database issue): D258-D261.
12. Kanehisa M. The KEGG database. Novartis Found Symp, 2002, 247: 91-101, 101-103, 119-128, 244-252.
13. Szklarczyk D, Gable AL, Lyon D, et al. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res, 2019, 47(D1): D607-D613.
14. Smoot ME, Ono K, Ruscheinski J, et al. Cytoscape 2. 8: new features for data integration and network visualization. Bioinformatics, 2011, 27(3): 431-432.
15. Chin CH, Chen SH, Wu HH, et al. CytoHubba: identifying hub objects and sub-networks from complex interactome. BMC Systems Biol, 2014, 8 Suppl 4(Suppl 4): S11.
16. Sood A, Granick MS, Tomaselli NL. Wound dressings and comparative effectiveness data. Adv Wound Care (New Rochelle), 2014, 3(8): 511-529.
17. Liu YC, Margolis DJ, Isseroff RR. Does inflammation have a role in the pathogenesis of venous ulcers: A critical review of the evidence. J Invest Dermatol, 2011, 131(4): 818-827.
18. Blumberg SN, Maggi J, Melamed J, et al. A histopathologic basis for surgical debridement to promote healing of venous ulcers. J Am Coll Surg, 2012, 215(6): 751-757.
19. Filkor K, Németh T, Nagy I, et al. The expression of inflammatory cytokines, TAM tyrosine kinase receptors and their ligands is upregulated in venous leg ulcer patients: a novel insight into chronic wound immunity. Int Wound J, 2016, 13(4): 554-562.
20. Crawford JM, Lal BK, Durán, WN, et al. Pathophysiology of venous ulceration. J Vasc Surg Venous Lymphati Disord, 2017, 5(4): 596-605.
21. Chin JS, Madden L, Chew SY, et al. Drug therapies and delivery mechanisms to treat perturbed skin wound healing. Adv Drug Deliv Rev, 2019, 149-150: 2-18.
22. Tian Y, Li H, Gao Y, et al. Quantitative proteomic characterization of lung tissue in idiopathic pulmonary fibrosis. Clin Proteomics, 2019, 16: 6.
23. Simka M, Rybak Z. Hypothetical molecular mechanisms by which local iron overload facilitates the development of venous leg ulcers and multiple sclerosis lesions. Med Hypotheses, 2008, 71(2): 293-297.
24. Okabe Y, Medzhitov R. Tissue biology perspective on macrophages. Nat Immunol, 2015, 17(1): 9-17.
25. Cairo G, Recalcati S, Mantovani A, et al. Iron trafficking and metabolism in macrophages: contribution to the polarized phenotype. Trends Immunol, 2011, 32(6): 241-247.
26. Wilkinson HN, Roberts ER, Stafford AR, et al. Tissue iron promotes wound repair via M2 macrophage polarisation and the chemokines CCL17 and CCL22. Am J Pathol, 2019, 189(11): 2196-2208.
27. Moghaddam AS, Mohammadian S, Vazini H, et al. Macrophage plasticity, polarization and function in health and disease. J Cell Physiol, 2018, 233(9): 6425-6440.
28. Yang HL, Tsai YC, Korivi M, et al. Lucidone promotes the cutaneous wound healing process via activation of the PI3K/AKT, Wnt/β-catenin and NF-κB signaling pathways. Biochim Biophys Acta Mol Cell Res, 2017, 1864(1): 151-168.
29. Weinheimer-Haus EM, Mirza RE, Koh TJ. Nod-like receptor protein-3 inflammasome plays an important role during early stages of wound healing. PLoS One, 2015, 10(3): e0119106.
30. Pukstad BS, Ryan L, Flo T H, et al. Non-healing is associated with persistent stimulation of the innate immune response in chronic venous leg ulcers. J Dermatol Sci, 2010, 59(2): 115-122.
31. Chen L, Guo S, Ranzer MJ, et al. Toll-like receptor 4 has an essential role in early skin wound healing. J Invest Dermatol, 2013, 133(1): 258-267.
32. Weyrich AS, Zimmerman GA. Platelets: signaling cells in the immune continuum. Trends Immunol, 2004, 25(9): 289-495.
33. Chiraz A, Fatma G, Dhafer L. Role of human macrophage polarization in inflammation during infectious diseases. Int J Mol Sci, 2018, 19(6): 1801.
34. Naik S, Larsen SB, Gomez NC, et al. Inflammatory memory sensitizes skin epithelial stem cells to tissue damage. Nature, 2017, 550(7677): 475-480.
35. Romagnani P, Lasagni L, Annunziato F, et al. CXC chemokines: the regulatory link between inflammation and angiogenesis. Trends Immunol, 2004, 25(4): 201-209.
36. Rennekampff HO, Hansbrough JF, Kiessig V, et al. Bioactive interleukin-8 is expressed in wounds and enhances wound healing. J Surg Res, 2000, 93(1): 41-54.
37. Schultz G, Wysocki A. Interactions between extracellular matrix and growth factors in wound healing. Wound Repair Regen, 2009, 17(2): 153-162.
38. Krishna NA, Pennington HM, Coppola CD, et al. Connecting local and global sensitivities in a mathematical model for wound healing. Bull Math Biol, 2015, 77(12): 2294-2324.
39. Tolg C, Liu M, Cousteils K, et al. Cell-specific expression of the transcriptional regulator RHAMM provides a timing mechanism that controls appropriate wound re-epithelialization. J Biol Chem, 2020, 295(16): 5427-5448.
40. Raffetto JD. Pathophysiology of wound healing and alterations in venous leg ulcers-review. Phlebology, 2016, 31(1 Suppl): 56-62.
41. Liew PX. The neutrophil's role during health and disease. Physiol Rev, 2019, 99(2): 1223-1248.
42. Mirastschijski U, Haaksma CJ, Tomasek JJ, et al. Matrix metalloproteinase inhibitor GM 6001 attenuates keratinocyte migration, contraction and myofibroblast formation in skin wounds. Exp Cell Res, 2004, 299(2): 265-275.
43. Prat C, Haas PJ, Bestebroer J, et al. A homolog of formyl peptide receptor-like 1 (FPRL1) inhibitor from Staphylococcus aureus (FPRL1 inhibitory protein) that inhibits FPRL1 and FPR. J Immunol, 2009, 183(10): 6569-6578.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Mining of differentially expressed genes of venous leg ulcers and screening of target genes

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