Keloid Tissue Research Articles

Efficient protocol for isolating human fibroblast from primary skin cell cultures: application to keloid, hypertrophic scar, and normal skin biopsies

Abstract This protocol introduces a streamlined and efficient method for isolating human fibroblast from skin primary cell culture with a specific focus on its application to keloid, hypertrophic scar, and normal skin biopsies. Additionally, the absence of suitable animal models for keloid and hypertrophic scar has led preclinical research to rely on in vitro studies using primary cell cultures. This approach addresses the challenges of existing protocols in terms of time, cost, equipment, and technical expertise required. The method involves derivation, culture, and characterization analysis including cell proliferation, migration, and fibroblastic marker (Vimentin, CD90, CD73, and CD105) expression. Our study yielded high amounts of fibroblast from tested skin explants while maintaining their in vivo-like characteristics and behaviour. Immunostaining assay confirmed that the cultivated fibroblast was positively expressed Vimentin. Flowcytometry results showed high expression of CD90 and CD73 while relatively showing lower expression of CD105. Fibroblast derived from keloid tissue showed the highest rate of proliferation and migration ability compared to the other samples. These findings suggest an efficient and reproducible technique to cultivate high qualified fibroblast from human skin in normal or pathological condition, particularly for keloid and hypertrophic scar. The application of this protocol provides a foundation for further studies to investigate the progression and potential intervention of aberrant fibrotic dermatological disorder, in vitro.

Analysis of subsets and localization of macrophages in skin lesions and peripheral blood of patients with keloids

Keloids are a type of abnormal fibrous proliferation disease of the skin, characterized by local inflammation that lacks clear pathogenesis and satisfactory treatment. The phenomenon of distinct phenotypes, including M1 and M2 macrophages, is called macrophage polarization. Recently, macrophage polarization has been suggested to play a role in keloid formation. This study aimed to evaluate the relation between macrophage polarization and keloids and identify novel effective treatments for keloids. Differentially expressed genes were identified via RNA sequencing of the skin tissue of healthy controls and patients with keloids and validated using quantitative PCR. Multiplex immunofluorescence microscopy was used to detect different phenotypes of macrophages in keloid tissues. Finally, quantitative PCR validation of differentially expressed genes and flow cytometry were used to analyze macrophages in the peripheral blood of healthy controls and patients with keloids. Total and M2 macrophages were significantly increased in the local skin tissue and peripheral blood of patients with keloids compared with healthy controls. In addition, inflammation- and macrophage polarization-related differentially expressed genes in keloid tissue showed similar expression patterns in the peripheral blood. This study highlighted an increased frequency of total macrophages and M2 polarization in the local skin tissue and peripheral blood of patients with keloids. This systematic macrophage polarization tendency also indicates a potential genetic predisposition to keloids. These findings suggest the possibility of developing new diagnostic and therapeutic indicators for keloids focusing on macrophages.

Identification of novel biomarkers of ferroptosis involved in keloid based on bioinformatics analysis.

Keloid is a fibroproliferative disease of unknown aetiology, which has a significant impact the quality of life of patients. Ferroptosis plays an important role in the occurrence and development of fibrosis, but there is still a lack of research related to keloids. The objective of this work was to identify the hub genes related to ferroptosis in keloid to better understand the keloid process. The microarray data (GSE7890 GSE145725, and GSE44270) (23 keloid and 22 normal fibroblast) were analysed via the gene expression comprehensive database (GEO). Only GSE7890 met the FerrDB database. Cell cycle and pathway analysis were performed with gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to differentially expressed genes (DEG). The differential genes were confirmed in other GEO datasets (GSE145725 and GSE44270), and multi-fibrosis-gene correlation analysed. To validate these hub genes, quantitative real-time PCR (qRT-PCR) was conducted. A total of 581 DEGs were screened, with 417 genes down-regulated and 164 genes up-regulated, with 11 ferroptosis genes significantly up-regulated in both keloid and normal tissue, and 6 genes are consistent with our findings and are associated with multiple fibrosis genes. The qRT-PCR results and tissues of normal skin and keloid agreed with our predictions. Our findings provide new evidence for the ferroptosis-related molecular pathways and biomarker of keloid.

Remdesivir alleviates skin fibrosis by suppressing TGF-β1 signaling pathway.

Fibrotic skin diseases, such as keloids, are pathological results of aberrant tissue healing and are characterized by overgrowth of dermal fibroblasts. Remdesivir (RD), an antiviral drug, has been reported to have pharmacological activities in a wide range of fibrotic diseases. However, whether RD function on skin fibrosis remains unclear. Therefore, in our study, we explored the potential effect and mechanisms of RD on skin fibrosis both in vivo and in vitro. As expected, the results demonstrated that RD alleviated BLM-induced skin fibrosis and attenuates the gross weight of keloid tissues in vivo. Further studies suggested that RD suppressed fibroblast activation and autophagy both in vivo and in vitro. In addition, mechanistic research showed that RD attenuated fibroblasts activation by the TGF-β1/Smad signaling pathway and inhibited fibroblasts autophagy by the PI3K/Akt/mTOR signaling pathway. In summary, our results demonstrate therapeutic potential of RD for skin fibrosis in the future.

Twist-related protein 1 promotes transforming growth factor β receptor 1 in keloid fibroblasts via regulating the stability of myocyte enhancer factor 2A.

Keloid scarring is caused by a fibroproliferative disorder due to abnormal activation of genes, the underlying mechanism of which is still unclear. The basic helix-loop-helix transcription factor Twist-related protein 1 (TWIST1) controls cell proliferation and differentiation in tissue development and disease processes. In this study, we aimed to clarify the essential role of TWIST1 in the pathogenesis of keloids. Immunohistochemistry, cell counting kit-8 assays, western blotting, PCR, matrigel invasion assays and immunofluorescence assays were applied to demonstrate the effects and mechanisms of TWIST1 in fibroblasts derived from normal skin and keloids. Mass spectrometry, ubiquitination assays, chromatin immunoprecipitation and dual luciferase reporter assay were applied to explore the interaction of TWIST1 with downstream molecules. In the present study, we confirmed that TWIST1 was upregulated in keloid tissue of patients and in keloid-derived fibroblasts (KFBs). In vitro, TWIST1 inhibition prevented KFB proliferation, invasion and activation. We also discovered a link between TWIST1 and the transforming growth factor β (TGF-β) signaling related molecules TGF-β receptor 1 (TΒR1), SMAD family member 2 (Smad2) and Smad3, and the fibrosis markers α-smooth muscle actin, collagen type I and collagen type III in KFBs. Mechanistically, we uncovered a brand-new mechanism by which TWIST1 interacts with myocyte enhancer factor 2A (MEF2A) and suppresses its ubiquitination and degradation. Using chromatin immunoprecipitation and dual-luciferase reporter assay, TΒR1 was identified as a novel downstream target of MEF2A, which directly binds to its promoter. Overexpression of TWIST1 promoted the recruitment of MEF2A to the TΒR1 promoter and restored TΒR1 functional expression. Our research highlights a significant function of TWIST1 in the development of keloid and its related fibroblasts, partially facilitated by elevated MEF2A-dependent TΒR1 expression. Blocking the expression of TWIST1 in KFBs could potentially pave a novel therapeutic avenue for keloid treatment.

Artificial keloid skin models: understanding the pathophysiological mechanisms and application in therapeutic studies.

Keloid is a type of scar formed by the overexpression of extracellular matrix substances from fibroblasts following inflammation after trauma. The existing keloid treatment methods include drug injection, surgical intervention, light exposure, cryotherapy, etc. However, these methods have limitations such as recurrence, low treatment efficacy, and side effects. Consequently, studies are being conducted on the treatment of keloids from the perspective of inflammatory mechanisms. In this study, keloid models are created to understand inflammatory mechanisms and explore treatment methods to address them. While previous studies have used animal models with gene mutations, chemical treatments, and keloid tissue transplantation, there are limitations in fully reproducing the characteristics of keloids unique to humans, and ethical issues related to animal welfare pose additional challenges. Consequently, studies are underway to create in vitro artificial skin models to simulate keloid disease and apply them to the development of treatments for skin diseases. In particular, herein, scaffold technologies that implement three-dimensional (3D) full-thickness keloid models are introduced to enhance mechanical properties as well as biological properties of tissues, such as cell proliferation, differentiation, and cellular interactions. It is anticipated that applying these technologies to the production of artificial skin for keloid simulation could contribute to the development of inflammatory keloid treatment techniques in the future.

Transcriptome network analysis of inflammation and fibrosis in keloids

BackgroundKeloid (KL) is a common benign skin tumor. KL is typically characterized by significant fibrosis and an intensive inflammatory response. Therefore, a comprehensive understanding of the interactions between cellular inflammation and fibrotic cells is essential to elucidate the mechanisms driving the progression of KL and to develop therapeutics. ObjectiveInvestigate the transcriptome landscape of inflammation and fibrosis in keloid scars. MethodsIn this paper, we performed transcriptome sequencing and microRNA (miRNA) sequencing on unselected live cells from six human keloid tissues and normal skin tissues to elucidate a comprehensive transcriptome landscape. In addition, we used single-cell RNA sequencing (scRNA-seq) analysis to analyze intercellular communication networks and enrich fibroblast populations in two additional keloid and normal skin samples to study fibroblast diversity. ResultsBy RNA sequencing and a miRNA-mRNA-PPI network analysis, we identified miR-615–5p and miR-122b-3p as possible miRNAs associated with keloids, as they differed most significantly in keloids. Similarly, COL3A1, COL1A2, THBS2, TNC, IGTA, THBS4, TGFB3 as genes with significant differences in keloid may be associated with keloid development. Using single-cell RNA sequencing data from 24,086 cells collected from normal or keloid, we report reconstructed intercellular signaling network analysis and aggregation to modules associated with specific cell subpopulations at the cellular level for keloid alterations. ConclusionsOur multitranscriptomic dataset delineates inflammatory and fibro heterogeneity of human keloids, underlining the importance of intercellular crosstalk between inflammatory cells and fibro cells and revealing potential therapeutic targets.

The HEDGEHOG-GLI1 pathway is important for fibroproliferative properties in keloids and as a candidate therapeutic target

Keloids are benign fibroproliferative skin tumors caused by aberrant wound healing that can negatively impact patient quality of life. The lack of animal models has limited research on pathogenesis or developing effective treatments, and the etiology of keloids remains unknown. Here, we found that the characteristics of stem-like cells from keloid lesions and the surrounding dermis differ from those of normal skin. Furthermore, the HEDGEHOG (HH) signal and its downstream transcription factor GLI1 were upregulated in keloid patient–derived stem-like cells. Inhibition of the HH-GLI1 pathway reduced the expression of genes involved in keloids and fibrosis-inducing cytokines, including osteopontin. Moreover, the HH signal inhibitor vismodegib reduced keloid reconstituted tumor size and keloid-related gene expression in nude mice and the collagen bundle and expression of cytokines characteristic for keloids in ex vivo culture of keloid tissues. These results implicate the HH-GLI1 pathway in keloid pathogenesis and suggest therapeutic targets of keloids.

Diagnosis and Treatment of Keloid: Method Summary and Effect Evaluation.

Keloid is a prevalent skin disorder characterized by the abnormal growth of keloid tissue, which usually occurs following wound healing or surgical incisions. It typically progresses through several stages: the inflammatory stage, the proliferative stage, collagen remodeling, and ultimately the formation of keloid. This review aims to summarize the diagnostic and therapeutic methods for keloid, and evaluate their effectiveness. The diagnosis of keloid is usually based on medical history and clinical manifestations such as pain, itching, erythema, and induration. Other commonly used diagnostic methods include tissue biopsy and ultrasound examination. Various treatment options for keloid exist, including physical therapy, medication, surgical treatment, and radiation therapy. Physical therapy includes pressure therapy, laser therapy, such as silicone sheets, elastic bandages, and laser irradiation. Medication treatment mainly involves the application of topical medications or intralesional injections, such as topical corticosteroids, 5-fluorouracil, and others. Radiation therapy can be administered using applicators and superficial radiation therapy, among other methods. The treatment outcomes of keloid vary from person to person and recurrence is common. Therefore, a comprehensive treatment approach may be the most effective strategy. Individualized treatment plans should consider factors such as the patient's age, gender, medical history, and the severity of the condition. In conclusion, the diagnosis and treatment of keloid require consideration of multiple factors and the implementation of individualized treatment plans. Future research should focus on identifying the molecular mechanisms underlying the occurrence and progression of keloid in order to develop more effective treatment methods.

68 Ga-FAPI-04 PET/CT in Assessment of Fibroblast Activation in Keloids : A Prospective Pilot Study.

Keloids are benign fibroproliferative disorders characterized by the massive proliferation of fibroblasts. Fibroblast activation plays a key role in the invasive growth of keloids. Therefore, a prospective pilot study was conducted to explore the value of 68 Ga-FAPI-04 PET/CT in the assessment of keloids activity. Twenty-five patients with keloid were enrolled to conduct 68 Ga-FAPI-04 PET/CT. All patients accepted surgery to remove part of the lesions within 1 week. SUV mean and SUV max were measured for semiquantitative analysis and compared with the Vancouver Scar Scale, Laser Speckle Contrast Imaging, pathology, and immunohistochemical stains. A total of 123 lesions were detected in 25 patients, most of which were distributed in the anterior chest wall. The 68 Ga-FAPI-04 uptake was significantly different at different sites ( P < 0.0001). There was uptake heterogeneity within the keloid lesions, and a significant difference was found between the edge and center of some large lesions. The SUV max of 68 Ga-FAPI-04 showed significantly correlation with the Vancouver Scar Scale ( r = 0.565, P < 0.0001) moderately and the Laser Speckle Contrast Imaging parameters mildly. The SUV max of 68 Ga-FAPI-04 had a moderate correlation with FAPI expression ( r = 0.520, P = 0.022). Moreover, collagen, fibroblast activator protein, and Ki-67 expression were found higher at the edges of keloid tissue than in the center. 68 Ga-FAPI-04 PET/CT can reflect the distribution characteristics of activated fibroblasts in keloid tissue and may provide a novel method for keloid evaluation for further fibroblast-related therapies.

The upregulation of circFoxp1 influences keloid by promoting cell proliferation.

As a result of abnormal wound healing in susceptible individuals, keloids are characterized by hyperproliferation of fibroblasts and excessive deposition of the extracellular matrix (ECM). Current surgical and therapeutic modalities provide limited satisfactory results. Circular ribonucleic acids (circRNAs) play a crucial role in the pathogenesis of various fibrotic diseases, but the potential biological function and expression profile of circRNAs in keloid formation remain unknown. In this study, we explored the function of circFoxp1 on keloid formation. Methods: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results revealed that circFoxp1 expression was higher in the keloid tissues. Furthermore, RNA-fluorescence in situ hybridization (RNA-FISH) and RNAscope illustrated that circFoxp1 was present in the cytoplasm. Subsequent cellular experiments demonstrated that circFoxp1 overexpression enhanced proliferation, migration, and ECM deposition. In addition, apoptosis was inhibited. Cell proliferation, inflammatory response, and oxidative phosphorylation of fibroblasts were also observed by RNA sequencing and were closely related to scar formation. The therapeutic potential of circFoxp1 was investigated by establishing keloid implantation models. In vivo, circFoxp1 can promote fibroblast proliferation and ECM deposition. RNA pull-down and western blot assays verified the interaction of circFoxp1 with RACK1. The present study reveals that circFoxp1 contributes to the pathological hyperplasia of keloid, which may improve inflammation and cell proliferation. Our data indicate that circFoxp1 may serve as a novel, promising therapeutic target, presenting a new avenue for understanding the underlying pathogenesis of keloid.

Weighted gene co-expression network analysis and machine learning identified the lipid metabolism-related gene LGMN as a novel biomarker for keloid.

The aetiology of keloid formation remains unclear, and existing treatment modalities have not definitively established a successful approach. Therefore, it is necessary to identify reliable and novel keloid biomarkers as potential targets for therapeutic interventions. In this study, we performed differential expression analysis and functional enrichment analysis on the keloid related datasets, and found that multiple metabolism-related pathways were associated with keloid formation. Subsequently, the differentially expressed genes (DEGs) were intersected with the results of weighted gene co-expression network analysis (WGCNA) and the lipid metabolism-related genes (LMGs). Then, three learning machine algorithms (SVM-RFE, LASSO and Random Forest) together identified legumain (LGMN) as the most critical LMGs. LGMN was overexpressed in keloid and had a high diagnostic performance. The protein-protein interaction (PPI) network related to LGMN was constructed by GeneMANIA database. Functional analysis of indicated PPI network was involved in multiple immune response-related biological processes. Furthermore, immune infiltration analysis was conducted using the CIBERSORT method. M2-type macrophages were highly infiltrated in keloid tissues and were found to be significantly and positively correlated with LGMN expression. Gene set variation analysis (GSVA) indicated that LGMN may be related to promoting fibroblast proliferation and inhibiting their apoptosis. Moreover, eight potential drug candidates for keloid treatment were predicted by the DSigDB database. Western blot, qRT-PCR and immunohistochemistry staining results confirmed that LGMN was highly expressed in keloid. Collectively, our findings may identify a new biomarker and therapeutic target for keloid and contribute to the understanding of the potential pathogenesis of keloid.

Identification of a potential bioinformatics-based biomarker in keloids and its correlation with immune infiltration

Keloid formation is a pathological consequence resulting from cutaneous irritation and injury, primarily attributed to excessive collagen matrix deposition and fibrous tissue proliferation. Chronic inflammation, left uncontrolled over an extended period, also stands as a substantial contributing factor. The precise mechanisms underlying keloid formation remain unclear. Therefore, this study aimed to identify key genes for diagnostic purposes. To achieve this, we used two Gene Expression Omnibus (GEO) data sets to identify differentially expressed genes. We identified one particular gene, homeobox C9 (HOXC9), using a thorough strategy involving two algorithms (least absolute shrinkage and selection operator and support vector machine-recursive feature elimination) and weighted gene co-expression network analysis. We then assessed its expression in normal and keloid tissues. In addition, we explored its temporal expression patterns via Mfuzz time clustering analysis. In our comprehensive analysis, we observed that immune infiltration, as well as cell proliferation, are crucial to keloid formation. Thus, we investigated immune cell infiltration in the keloid and normal groups, as well as the correlation between HOXC9 and these immune cells. It was found that HOXC9 was closely associated with the immune microenvironment of keloids. This shows that HOXC9 can serve as a potential biomarker and therapeutic target for keloids.

Inhibition of keloid by 32P isotope radiotherapy through suppressing TGF-β/Smad signaling pathway.

Keloid seriously affects the appearance, and is accompanied by some symptoms including pain, burning, itching. Radioactive nuclides such as 32P have been proved to be effective in inhibiting the formation of keloid, but the mechanism remains unclear. The keloid animal model was established through keloid tissues implantation. Hematoxylin-Eosin (HE) and Masson staining were performed to investigate histological changes and collagen deposition. The mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. Cell apoptosis and cycle were evaluated through flow cytometry. Both 32P isotope injection and skin path significantly reduced the size of keloid, and inhibited TGF-β/Smad signaling pathway. SRI-011381, the agonist of TGF-β/Smad signaling pathway, markedly reversed the influence of 32P isotope on cell proliferation, cell apoptosis, cell cycle of LNCaP cells and TGF-β/Smad signaling pathway. 32P isotope injection and skin path greatly reduced the size of keloid, and the TGF-β/Smad signaling pathway was remarkably inhibited by 32P isotope treatment. The regulation of dermal fibroblast by 32P isotope was reversed by SRI-011381. 32P isotope might inhibit keloid through suppressing TGF-β/Smad signaling pathway. Our study provides a novel therapeutic strategy for the treatment of keloid.

ZC3H13 Accelerates Keloid Formation by Mediating N6-methyladenosine Modification of HIPK2.

Keloids are fibroproliferative skin disorders caused by the improper healing of wounded skin. A growing body of evidence suggests the involvement of N6-Methyladenosine (m6A) modification in various bioprocesses; however, its role in keloid formation has not yet been investigated. The aim of this study was to determine the effect of the m6A regulator zinc finger CCCH domain containing protein 13 (ZC3H13) on the pathogenesis of keloid formation. ZC3H13 and homeodomain-interacting protein kinase 2 (HIPK2) expression was evaluated in healthy skin and keloid tissues, as well as in human dermal fibroblasts and human keloid fibroblasts (HKF), using qRT-PCR and western blotting. The effects of ZC3H13 overexpression and knockdown on the cell function of HKFs were assessed using CCK8, transwell, and flow cytometry. Furthermore, the influence of ZC3H13 on HIPK2 m6A modification was assessed using MeRIP-qPCR and mRNA stability assays. Both ZC3H13 expression and m6A RNA methylation were upregulated in keloid tissues and HKFs. Silencing of ZC3H13 inhibited proliferation and migration, while enhancing apoptosis in HKFs, whereas overexpression had the opposite effect. Furthermore, HIPK2 levels were high in keloid tissues and HKFs, and a positive correlation was observed between ZC3H13 and HIPK2. In HKFs, ZC3H13 overexpression elevated the m6A levels of HIPK2 mRNA and reduced the rate of HIPK2 mRNA degradation. Mechanically, ZC3H13-induced m6A modifications significantly improved HIPK2 mRNA stability. Collectively, ZC3H13 accelerated keloid formation by mediating the m6A modification of HIPK2 mRNA and maintaining its stability.

The biomarkers associated with epithelial-mesenchymal transition in human keloids

IntroductionA keloid is a type of benign fibrotic disease with similar features to malignancies, including anti-apoptosis, over-proliferation, and invasion. Epithelial-mesenchymal transition (EMT) is a crucial mechanism that regulates the metastatic behavior of tumors. Thus, identifying EMT biomarkers is paramount in comprehensively understanding keloid pathogenesis. MethodsTo identify the differentially expressed genes (DEGs) GSE92566 dataset, with 3 normal skin and 4 keloid tissues, was downloaded from GEO databases to identify the differentially expressed genes (DEGs). Further, EMT-related genes were downloaded from dbEMT 2.0 databases and intersected with GSE92566 DEGs to identify EMT-related-DEGs (ERDEGs). Subsequently, the ERDEGs were used for GO, KEGG, gene set enrichment analysis (GSEA), protein-protein interaction (PPI), and miRNAs-mRNAs network analysis. To predict small molecules for EMT inhibition, the ERDEGs were imported to cMAP databases, whereas hub genes were imported to DGidb databases. Finally, we carried out qRT-PCR and in vitro experiments to validate our findings. ResultsA total of 122 ERDEGs were identified, including 59 upregulated and 63 down-regulated genes. Moreover, enrichment analysis revealed that focal adhesion, AMPK signal pathway, Wnt signal pathway, and EMT biological process were significantly enriched. STRING databases and Cytoscape software were used to construct the PPI network and EMT-related hub genes. Further, 3 modules were explored from the PPI network using the Molecular Complex Detection (MCODE) plugin. In the Cytohubba plugin, 10 hub genes were explored, including FN1, EGF, SOX9, CDH2, PROM1, EPCAM, KRT19, ITGB1, CD24, and KRT18. These genes were then enriched for the focal adhesion pathway. We constructed a microRNA (miRNA)-mRNA network, which predicted hsa-miR-155–5p (8 edges), hsa-miR-124–3p (7 edges), hsa-miR-145–5p (5 edges), hsa-miR-20a-5p (5 edges) and hsa-let-7b-5p (4 edges) as the most connected miRNAs regulating EMT. Based on the ERDEGs and 10 hub genes mentioned above, ribavirin demonstrated high drug-targeting relevance. Subsequently, qRT-PCR confirmed that the expression of FN1, ITGB1, CDH2, and EPCAM corroborated with previous findings. qRT-PCR also showed that the expression levels of hsa-miR-124–3p and hsa-miR-145–5p were significantly lower in keloids and hsa-miR-155–5p was upregulated in keloids. Finally, by treating human keloid fibroblasts (HKFs) with ribavirin in vitro, we confirmed that ribavirin could inhibit HKFs proliferation and EMT. ConclusionIn summary, this work provides novel EMT biomarkers in keloids and predicts new small target molecules for keloid therapy. Our findings improve the understanding of keloid pathogenesis, providing new treatment options.

Experimental Study of Warburg Effect in Keloid Nodules: Implication for Downregulation of miR-133b.

A keloid is composed of several nodules, which are divided into two zones: the central zone (CZ; a hypoxic region) and the marginal zone (MZ; a normoxic region). Keloid nodules play a key role in energy metabolic activity for continuous growth by increasing in number and total area. In this study, we aimed to investigate the roles of the zones in the execution of the Warburg effect and identify which microRNAs regulate this phenomenon in keloid tissue. Eleven keloids from patients were used. Using immunohistochemical analysis, 179 nodules were randomly chosen from these keloids to identify glycolytic enzymes, autophagic markers, pyruvate kinase M (PKM) 1/2, and polypyrimidine tract binding protein 1 (PTBP1). Western blot and qRT-PCR tests were also performed for PKM, PTBP1, and microRNAs (miR-133b and miR-200b, c). Immunohistochemical analysis showed that the expression of the autophagic (LC3, p62) and glycolytic (GLUT1, HK2) were significantly higher in the CZ than in the MZ. PKM2 expression was significantly higher than PKM1 expression in keloid nodules. Furthermore, PKM2 expression was higher in the CZ than in the MZ. However, PKM1 and PTBP1 expression levels were higher in the MZ than in the CZ. The qRT-PCR analysis showed that miR-133b-3p was moderately downregulated in the keloids compared with its expression in the normal skin tissue. The Warburg effect occurred individually in nodules. The MZ presented PKM2-positive fibroblasts produced by activated PTBP1. In the CZ, PKM2-positive fibroblasts produced lactate. MiR-133b-3p was predicted to control the Warburg effect in keloids.

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue.

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and developmentof keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFRα and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

Hsa_circ_0000129 knockdown attenuates proliferation and migration in keloid fibroblasts by targeting miR-485–3p/SGMS2 pathway

BackgroundAberrant biofunction of circular RNAs (circRNAs) is potently implicated in keloid formation. However, their roles have been underinvestigated. Recent evidence has demonstrated the pro-tumor role of circ_0000129 in cancers, and yet its role in keloid remains elusive. MethodsRT-qPCR analysis and or western blotting of miR-485–3p, circ_0000129, and SGMS2 in keloid tissues and keloid fibroblasts was implemented. CCK8, EdU, scratch wound healing, and Transwell migration assays were perfomed to determine the keloid fibroblast proliferation and migration. Luciferase reporter and RIP assays were adopted to analyze the interaction among circ_0000129, miR-485–3p and SGMS2. ResultsIn keloid tissues and keloid fibroblasts, circ_0000129 and SGMS2 were amplified, although miR-485–3p expression was downregulated. Furthermore, siRNAs-targeting endogenous circ_0000129 resulted in proliferation and migration defect of keloid fibroblasts. MiR-485–3p was simultaneously recognized by circ_0000129 and SGMS2 3′UTR. Rescued functional assays also illustrated that miR-485–3p loss was beneficial to the proliferation and migration of keloid fibroblasts, and these promoting changes were nullified by accompanied silence circ_0000129 or SGMS2. ConclusionCirc_0000129 sponges miR-485–3p and releases expression of SGMS2 from the miR-485–3p suppression, promoting migration and proliferation of keloid fibroblasts, suggesting targeting circ_0000129/miR-485–3p/SGMS2 might be a promising strategy against keloid fibroblasts. Availability of data and materialAll data generated or analyzed during this study are included in this article.

Role and mechanism of Circ-PDE7B in the formation of keloid.

The excessive proliferation of keloid fibroblasts is one of the important reasons leading to the formation of keloids. Circular RNA (circRNA) is an important regulator that regulates the biological functions of cells. However, the role and mechanism of circ-PDE7B in keloid formation have not been studied yet. QRT-PCR was used to detect the circ-PDE7B, miR-331-3p and cyclin-dependent kinase 6 (CDK6) expression. The biological functions of keloid fibroblasts were determined by MTT assay, flow cytometry, transwell assay and wound healing assay. Western blot analysis was used to measure the protein levels of extracellular matrix (ECM) markers and CDK6. The interaction between miR-331-3p and circ-PDE7B or CDK6 was confirmed by dual-luciferase reporter assay and RIP assay. Circ-PDE7B was found to be upregulated in keloid tissues and fibroblasts. Downregulation of circ-PDE7B could suppress the proliferation, invasion, migration, ECM accumulation and accelerate the apoptosis of keloid fibroblasts. Circ-PDE7B could serve as a sponge of miR-331-3p, and the regulation of silenced circ-PDE7B on the biological functions of keloid fibroblasts could be abolished by miR-331-3p inhibitor. Additionally, CDK6 was a target of miR-331-3p, and its overexpression could reverse the negative regulation of miR-331-3p on the biological functions of keloid fibroblasts. Circ-PDE7B sponged miR-331-3p to positively regulate CDK6 expression. Taken together, circ-PDE7B promoted the proliferation, invasion, migration and ECM accumulation of keloid fibroblasts by regulating the miR-331-3p/CDK6 axis, suggesting that circ-PDE7B might be a potential target for keloid treatment.