| Objectives:Lichen sclerosus(LS)is an immune-mediated chronic inflammatory skin mucosal lesion that occurs in the genital skin,with extra-genital involvement being rare.The pathogenesis of this disease is unknown,and it can affect the quality of life of patients,has a risk of malignancy,and is easily misdiagnosed when the clinical presentation is atypical.Single-cell RNA sequencing(scRNA-seq)technology can create an individual transcriptome library for each cell and cluster it into different cell populations to obtain its full transcriptome expression profile in the disease,thus revealing the molecular regulatory mechanisms in the development of the disease.Currently,single-cell-based molecular alterations in LS are largely unknown.This project will focus on classifying the cells available in vulva lichen sclerosus(VLS)lesions by single-cell transcriptome sequencing,constructing cell population profiles of VLS and normal vulvar skin tissues,and analyzing gene expression based on cell type The analysis was performed to assess the differences in transcriptome expression and biological functions of upregulated genes in VLS tissues,in an attempt to provide new ideas for the treatment of VLS.In addition,this study will also analyze the clinical and pathological features of progressive genital and extragenital LS and their differences,with the aim of improving clinicians’understanding of the disease and reducing misdiagnosis.Methods:1.We retrospectively analyzed the clinicopathological data of 55 patients with progressive genital and extragenital LS diagnosed by biopsy in the dermatology department of Beijing Hospital and compared the differences.2.We collected vulvar skin specimens from two VLS patients and two healthy people,prepared cell suspensions,and performed scRNA-seq.Unsupervised clustering of cells from all samples was performed using Seurat software,and its FindClusters function was used to find significantly different genes for each cell cluster,from which marker genes for each cluster were screened,and thus each cell type was defined;Wilcoxon rank sum test was performed with Seurat software for the proportion of each cell type to all cell types in each sample.The Wilcoxon rank sum test for two independent samples was performed with Seurat software for the ratio of each cell type to all cell types in each sample between the VLS group and the control group;after grouping the cells according to the samples and subgroups to which they belonged,the FindMarkers function was used to analyze the genes that were upregulated in the VLS group compared to the control group within the same cluster of cell groups mainly involved in the pathological mechanism of VLS.The differentially expressed genes were analyzed by using the FindMarkers function.Combining the indicators pct.1,pct.2 and avg_log2 FC with the clinicopathological and molecular biological characteristics of VLS,we screened the genes most likely to be related to the pathological mechanism of VLS and Disease Ontology(DO),Gene Onotology Consortium(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis was performed to explore their related functions.Results:1.The proportion of patients with asymptomatic extragenital LS was significantly higher than that of extragenital LS,the proportion of genital LS with pruritus was significantly higher than that of extragenital LS,and pain was seen only in genital LS.The proportion of genital LS specimens with acantholytic hypertrophy,vasodilatation,and eosinophilic infiltration was higher than that of extragenital LS,and the proportion of follicular keratosis was lower than that of extragenital LS.2.A total of 41090 cells were examined,including 18467 in the VLS group and 22623 in the control group.11 cell populations were mainly distinguished based on marker gene annotation,including keratinocytes(19325,44.6%),endothelial cells(6747,15.6%),fibroblasts(5718,13.2%),T cells(3497,8.1%),smooth muscle cells(2228,5.1%),melanocytes(1138,2.6%),dendritic cells(807,1.9%),mast cells(631,1.5%),macrophages(570,1.3%),neuronal cells(237,0.5%),and plasma cells(192,0.4%).3.A total of 32 differentially expressed genes were upregulated in keratinocytes of VLS lesions,and the most likely genes associated with VLS pathogenesis were CXCL14,SAA1,and ADGRL.DO enrichment indicated that the upregulated genes may be primarily associated with herpetic dermatosis,GO functional analysis showed that these genes were primarily enriched in genes related to cell-extracellular matrix adhesion junctions,regulation of fibroblast proliferation biological functions,keratin filaments,and intermediate filament cytoskeleton,and KEGG enrichment showed that these genes may be associated with Notch signaling pathway,antigen processing and presentation,melanin formation,neurotrophin signaling pathway,Wnt signaling pathway,calcium signaling pathway,and chemokine signaling pathway.This may indicate that the upregulated genes in this group mainly affect the composition of the VLS cytoskeleton and regulate the proliferation of fibroblasts by regulating related signaling pathways.4.A total of 8 differentially expressed genes were upregulated in vascular endothelial cells from VLS lesions,and the most likely gene associated with the pathogenesis of VLS was ID3.DO enrichment indicated that the upregulated genes may be associated with ulcerative colitis,and GO and KEGG enrichment showed that these genes were mainly enriched in the negative regulation of leukocyte migration,calcium ion release,melanin formation,TGF-β signaling pathway,calcium signaling pathway,chemokine signaling pathway,and Wnt signaling pathway.It is hypothesized that the upregulated genes in this cluster may regulate the inflammatory response in VLS tissues by regulating related signaling pathways and thus affecting leukocyte migration and cell activation,proliferation,and promoting tissue repair.5.A total of 25 differentially expressed genes were upregulated in fibroblasts from VLS lesions,and the most likely genes associated with the pathogenesis of VLS were CLU,IGFBP,and COL6A3.DO enrichment indicated that the upregulated genes may be associated with lung cancer,and GO and KEGG enrichment showed that these genes were mainly enriched in connective tissue development,extracellular components of collagen composition,antigen processing and presentation,chemokine signaling pathway,mTOR signaling pathway,P53 signaling pathway,and Toll-like receptor signaling pathway.This may indicate that the upregulated genes in this cluster mainly affect VLS cell growth and proliferation,extracellular matrix construction and connective tissue development by regulating related signaling pathways.6.69 up-regulated genes were screened in T-cells of VLS lesions,and the most likely genes associated with the pathogenesis of VLS were PIP,TSHZ2,and PDE3B.Upregulated genes may be mainly associated with lymphocytic leukemia,autoimmune diseases,tuberculosis,etc.The main focus of these genes is on the regulation of local adhesion components,regulation of cell-matrix junction components,antigen processing and presentation,phosphatidylinositol signaling system,and neuroactive ligand-receptor interactions.It is hypothesized that this group of highly expressed genes may affect the phosphatidylinositol signaling system and neural activity through the regulation of cellcell and cell-extracellular matrix local adhesions,which are associated with the development of VLS.7.A total of 39 up-regulated genes were screened in melanocytes from VLS lesions,and the most likely genes associated with the pathogenesis of VLS were CD74,HLADRB1,and HLA-DRA.Up-regulated genes may be mainly associated with hepatitis,autoimmune diseases(multiple sclerosis,Graves’ disease,type 1 diabetes,etc.).These genes are mainly enriched in IFN-γ-mediated signaling pathway,antigen processing and delivery,Toll-like receptor signaling pathway,Jak-STAT signaling pathway,and chemokine signaling pathway.It is hypothesized that this group of highly expressed genes in VLS may be mainly involved in antigen processing and delivery,regulation of IFN-ymediated signaling pathways,Toll-like receptor signaling pathways,and thus in antimicrobial and regulatory inflammatory responses.8.A total of 66 up-regulated genes were screened in macrophages from VLS lesions,and the most likely genes associated with the pathogenesis of VLS were CXCL10,FN1,and CCL18.Up-regulated genes may be mainly associated with lung disease.These were mainly enriched in defense response to virus,antigen processing and delivery,Jak-STAT signaling pathway,chemokine signaling pathway,cell-cytokine receptor interaction,Tolllike receptor signaling pathway,etc.It is hypothesized that the main roles of this group of highly expressed genes in VLS are related to the processing of presented antigens,resistance to pathogens,and regulation of immune responses.9.A total of 38 up-regulated genes were screened in dendritic cells from VLS lesions,and the most likely genes associated with the pathogenesis of VLS were IGKC,CXCL9,and DCD.Up-regulated genes may be associated with atopic dermatitis.These differential genes were mainly enriched in humoral immune response,chemokine activity,cytokine-cytokine receptor interactions,and Toll-like receptor signaling pathway.It is hypothesized that this group of upregulated genes may mainly play an anti-infective and regulatory immune response role together with melanocytes and macrophages.10.A total of 22 up-regulated genes were screened in mast cells of VLS lesions,and the most likely genes associated with the pathogenesis of VLS were MUCL1,PIP,and HES1.Up-regulated genes may be mainly associated with lymphocytic leukemia.These genes were mainly enriched in the regulation of glial cell proliferation and chemokine production,antigen processing and presentation,Notch signaling pathway,Toll-like receptor signaling pathway,and chemokine signaling pathway.It is hypothesized that the high expression of these genes may affect the development of the disease mainly by influencing the proliferation of neuronal cells and the production of chemokines in VLS.Conclusions:1.There are some differences between progressive genital LS and extragenital LS in clinical symptoms and pathological changes such as follicular keratosis,acantholytic hypertrophy,vasodilatation and eosinophilic infiltration.2.Keratinocytes,endothelial cells,fibroblasts,T cells,melanocytes,macrophages,dendritic cells,and mast cells in VLS lesion tissues have differentially expressed genes that are upregulated compared to normal skin.3.VLS may be associated with herpetic dermatoses,ulcerative colitis,lung cancer,lymphocytic leukemia,autoimmune diseases,hepatitis,and atopic dermatitis.4.Fibroblasts,melanocytes,macrophages,dendritic cells,and mast cells in VLS may be involved in the regulation of Toll-like receptor signaling pathways,thereby affecting the production of pro-inflammatory factors,anti-inflammatory factors,and chemokines to cause local inflammation and modulate immune response. |
PDF Full Text Request