Identification and Verification of SLC6A15 Involved in Keloid via Bioinformatics Analysis and Machine Learning.

Mounting evidence has reported that microRNAs (miRNAs) play irreplaceable roles in the development of keloid fibrosis. miR-4417 has been reported to contribute to nickel chloride-promoted lung epithelial cell fibrogenesis and tumorigenesis. However, whether miR-4417 is involved in keloid fibrogenesis as well as its underlying mechanisms remain largely elusive. In this study, the expression levels of miR-4417 and CyclinD1 in keloid tissues and fibroblasts were examined by qRT-PCR. Cell proliferation was determined by CCK assay. Western blot and flow cytometry were performed to evaluate cell apoptosis. Cell migration and invasion were measured by Transwell assay. Luciferase reporter assay was used to confirm the relationship between miR4417 and CyclinD1. As a result, we found that miR-4417 was significantly down-regulated in keloid tissues and fibroblasts. miR-4417 up-regulation led to the suppression of proliferation, migration, and invasion, while induced cell apoptosis in keloid fibroblasts. However, miR-4417 depletion exerted an opposite effect. CyclinD1 harbored the binding sites with miR-4417. Besides, the expression of CyclinD1 was evidently decreased in keloid tissues and fibroblasts. Meanwhile, miR-4417 was negatively correlated with CyclinD1 in keloid tissue. The effect of CyclinD1 knockdown on keloid fibroblasts was similar to that of miR-4417 overexpression. Furthermore, the elevated of CyclinD1 expression rescued the effect of miR-4417 up-regulation on keloid fibroblasts. miR-4417/CyclinD1 axis was required for cell proliferation, apoptosis, migration, and invasion in keloid fibroblasts. In conclusion, miR-4417 and CyclinD1 may be potential therapeutic targets for the treatment of keloid.

Keloid is characterized by excessive collagen accumulation and fibroblast growth, which are fibroproliferative disorders of injured skin, causing functional limitations. Studies have shown that adipose-derived stem cells (ADSCs) inhibit the bioactivity and fibrosis of keloid fibroblasts. However, the molecular mechanism of this effect of ADSCs on keloid formation has not been fully elucidated. This in vitro study used fibroblasts obtained from keloids. A consensus gene co-expression network was constructed to focus on identifying consensus gene co-expression modules associated with keloid fibroblasts. Differentially expressed genes (DEGs) were identified between keloid fibroblasts and normal dermal fibroblasts. A functional enrichment analysis was also performed with the DAVID database. A weighted gene co-expression network analysis (WGCNA) was used to screen keloid-related modules using the "WGCNA" R package, followed by hub gene selection in modules from the Protein-protein interaction network through the STRING database. Keloid fibroblasts and ADSCs were extracted and cultured. Proliferation and apoptosis were examined using a 5-ethynyl-2-deoxyuridine (Edu) kit and flow cytometry. We identified 302 DEGs overlapping with a consensus analysis of clusters and a differential expression analysis between keloid fibroblasts and normal dermal fibroblasts. Most of these were involved in collagen binding, extracellular matrix organization, and the PI3K-Akt signaling pathway. WGCNA analysis selected a keloid-associated brown module. ITGA2 was identified as a novel marker in hub genes from the PPI network based on the degree and function of collagen modulation. Furthermore, the proliferation ability of keloid fibroblasts cultured in ADSC medium was inhibited while apoptosis was dramatically increased. Overexpression of ITGA2 reversed the decrease in ADSC-induced apoptosis and increased ADSC-reduced proliferation. Our study demonstrated that activation of ITGA2 plays a crucial role in ADSC-induced keloid fibroblast apoptosis and anti-proliferation effects. These results also improved our understanding of the molecular mechanism of the pathogenesis of keloid in response to ADSCs and may contribute to the further development of keloid therapy.

Keloid disease is a fibroproliferative disorder, with an ill-defined treatment that is characterized by excessive extracellular matrix deposition. Mechanical tension promotes deposition of extracellular matrix and overexpression of tension-related proteins, which is associated with keloid disease. The aim of this study was to investigate the effect of tension-related proteins on extracellular matrix steady-state synthesis in primary keloid fibroblasts. Keloid fibroblasts (n = 10) and normal skin (n = 4) fibroblast cultures were established from passages 0 to 3. A panel of 21 tension-related genes from microarray data were assessed at mRNA (quantitative reverse-transcriptase polymerase chain reaction) and protein (in-cell Western blotting) levels. Three genes were significantly altered in keloid tissue and fibroblasts, and their functional role was assessed using siRNA knockdown. Hsp27, α2β1-integrin, and PAI-2 were significantly up-regulated (p < 0.05)in keloid tissue and fibroblasts compared with normal skin. Hsp27, α2β1-integrin, and PAI-2 expression was inhibited by RNA interference. Both the mRNA and protein levels of Hsp27, α2β1-integrin, and PAI-2 significantly decreased (p < 0.05) in keloid fibroblasts at 48 hours after transfection. After down-regulation of Hsp27, α2β1-integrin, and PAI-2, the expression of intracellular extracellular matrix was significantly reduced (p < 0.05). Water-soluble tetrazolium salt-1 assay showed that transfection of Hsp27, α2β1-integrin, and PAI-2 did not influence the viability/metabolic activity of keloid fibroblasts. This study demonstrates overexpression of key tension-related proteins in keloid tissue and keloid fibroblasts. Knockdown of Hsp27, PAI-2, and α2β1-integrin by RNA interference attenuates the expression of mRNA and protein levels and certain other extracellular matrix molecules.

Background/Aims: Keloids are fibrous overgrowths induced by cutaneous injury. MicroRNAs (miRNAs) have recently emerged as post-transcriptional gene repressors and participants in a diverse array of pathophysiological processes leading to skin disease. The purpose of the current study was to explore the precise functions of miR-181a in human keloid development and the underlying mechanisms. Methods: A miRNA microarray analysis was performed to compare expression profiles between keloid and normal skin tissues. Quantitative real-time PCR was conducted to estimate miR-181a expression. Cell proliferation was determined using the cell counting kit-8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU) assays, and cell cycle and apoptosis were detected with flow cytometry. Direct targets of miR-181a were identified using the luciferase reporter assay. Results: miR-181a was significantly upregulated in human keloid tissues and fibroblasts, compared with their control counterparts. Overexpression of miR-181a enhanced keloid fibroblast DNA synthesis and proliferation and inhibited apoptosis, whereas miR-181a suppression triggered the opposite effects. Moreover, miR-181a suppressed the expression of PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) through direct interactions with its 3′UTR region and subsequently enhanced AKT activation. Overexpression of PHLPP2 without its 3′UTR attenuated the effects of miR-181a on cell proliferation and apoptosis in keloid fibroblast cells. Furthermore, miR-181a mimics increased normal skin fibroblast proliferation. Conclusions: Our results highlight a novel pathway mediated by miR-181a, which may be effectively used as a therapeutic target for treatment of keloids.

To explore the effect of simvastatin on the proliferation, apoptosis and protein expressions of keloid fibroblasts under normoxia,hypoxia or TGF-β1 treatment. Keloid fibroblasts (KFs) were isolated by explants culture method. KFs were treated with different concentrations of simvastatin under normoxia or hypoxia (2％ O2) for 24 h and 48 h. The effects of simvastatin on cell proliferation were detected by CCK-8.Flow cytometer was used to detect the apoptosis of KFs treated with 10 μ mol/L simvastatin for 24 h or 48 h under normoxia, hypoxia or 10 ng/ml TGF-β1 treatment. Then the expressions of keloid-related proteins were analyzed by Western Blot. It showed that simvastatin could inhibit the proliferation of KFs in a concentration-and time-dependent manner with the concentration range of 10-500 μ mol/L for 24 h and 0.1-500 μ mol/L for 48 h. This inhibitory effect could be significantly enhanced when cells were incubated under hypoxia for 48h with 10-500 μ mol/L simvastatin.10 μ mol/L simvastatin could not influence the apoptosis of KFs under normoxia or TGF-β1 treatment, neither incubated for 24 h nor 48 h.When incubated under hypoxia,10 μ mol/L simvastatin could significantly induce the apoptosis of KFs, with the rate of 155.6％ for 24 h and 478.8％ for 48 h, compared with no-drug control. There are no significant influences on the expression of type Ⅰ collagen, CTGF or TIMP-1 when KFs were treated with 10 μ mol/L simvastatin under normoxia for 48 h. When incubated with 10 ng/ml TGF-β1 together with 10 μmol/L simvastatin for 48 h, the expression of CTGF was significantly inhibited. KFs treated with 10 μ mol/L simvastatin under hypoxia for 48 h showed a significant decrease of type Ⅰ collagen and CTGF, and a significant increase of TIMP-1. Simvastatin has different effects on the proliferation, apoptosis and protein expressions of KFs in a dosedependent manner under different conditions. The effects are enhanced under hypoxia.

Keloids are considered to be a type of benign tumor. MicroRNAs have been reported to be involved in the formation and growth of keloids. MicroRNA-4328 (miR-4328) was found to be abnormally expressed in keloids, while the role and the detailed molecular mechanism of miR-4328 in keloids remain unclear. The expression of miR-4328 and B-cell lymphoma 2 (BCL2) mRNA was detected by qRT-PCR. The proliferation, migration, invasion and apoptosis of keloid fibroblasts (KFs) was examined using Cell Counting Kit-8 assay, transwell assay or flow cytometry, respectively. Western blot was used to detect the level of proliferating cell nuclear antigen, cleaved-caspase 3, collagen I, collagen III and BCL2 protein. The interaction between miR-4328 and BCL2 was confirmed by luciferase reporter analyses. It was observed that miR-4328 was down-regulated in keloid tissues and fibroblasts, and miR-4328 restoration mediated the inhibition of proliferation, metastasis, collagen synthesis and the promotion of apoptosis in KFs. BCL2 was up-regulated in keloid tissues and fibroblasts, and BCL2 knockdown promoted the deterioration of KFs. In addition, BCL2 was confirmed to be a target of miR-4328, and the rescue experiment indicated that the inhibitory action of miR-4328 on keloid fibroblast progression was reversed by BCL2 overexpression. Thus, our results demonstrated that miR-4328 restrained the deterioration of KFs by targeting BCL2, which sheds new light on miR-4328 as a promising target for keloid development and therapeutic.

CircCOL5A1 inhibits proliferation, migration, invasion, and extracellular matrix production of keloid fibroblasts by regulating the miR-877-5p/EGR1 axis

Keloids are considered benign dermal fibroproliferative tumors. Keloid fibroblasts (KFs) persistently proliferate and fail to undergo apoptosis, and no treatment is completely effective against these lesions. Tanshinone IIA induces apoptosis and inhibits the proliferation of various tumor cell types. In this study, we investigated the effect of tanshinone IIA on the regulation of proliferation, cell cycle, and apoptosis in KFs, and investigated potential mechanisms involved in the effects. First, KFs and normal skin fibroblasts (NSFs) were treated with various concentrations of tanshinone IIA. Cell counting kit-8 (CCK-8) was used to assess the proliferative activity of KFs and NSFs, and flow cytometry was used to investigate the cell cycle and apoptosis in KFs. We found that the proliferation of all tanshinone IIA-treated KFs was significantly decreased after treatment for 72 hours (P < 0.001). Also, NSFs treated with tanshinone IIA did not exhibit noticeable effects compared with KFs. In addition, the percentages of G0/G1 cells in all tanshinone IIA-treated KFs were significantly increased after treatment for 72 hours (P < 0.001). And the percentages of cells undergoing early apoptosis in all tanshinone IIA-treated KFs were significantly increased after treatment for 120 hours (P < 0.001). Furthermore, the apoptosis antibody array kit and Western blot analysis revealed that tanshinone IIA decreased survivin expression in KFs (P < 0.001). In conclusion, tanshinone IIA downregulates survivin and deactivates KFs, thus suggesting that tanshinone IIA could serve as a potential clinical keloid treatment.

To characterize apoptosis in keloids and the mechanisms responsible for this process, the expression of activated caspase-9 and -3 in fibroblasts obtained from keloids was analyzed. Immunohistochemistry revealed that the number of fibroblasts positive for terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) or activated caspase-9 or -3 was low but was significantly higher in keloid tissues than in normal scar tissues. Significant relationships between the number of caspase-positive fibroblasts and TUNEL-positive fibroblasts suggested that the activation of caspase-9 and -3 induces apoptosis in a subpopulation of keloid fibroblasts. All keloid fibroblast cell lines established in this study showed activation of caspase-9 and -3 after serum deprivation for 3 or 4 hours, as shown using Western blotting. Furthermore, serum deprivation-induced apoptosis in a keloid fibroblast line was blocked by a caspase-9 inhibitor (acetyl-Leu-Glu-His-Asp-al), indicating that activation of caspase-9 was necessary for the process of apoptosis in keloid fibroblasts. Although serum deprivation did not significantly change the level of apoptosis protease activating factor-1 in any of the lines, cytochrome c release was detected in cytosolic fractions of the lines after serum deprivation for 3 or 4 hours. These results strongly suggest that keloid fibroblasts are predisposed to apoptosis and cytochrome c release and that caspase-9 activation may underlie regulation of apoptosis in keloid fibroblasts in vivo.

The excessive growth of fibroblasts in keloid is closely related to the status of gene methylation. The aim of this project was to study whether keloid development is related to DNA methylation in the CDC2L1 gene promoter region. DNA methylation of the promoter of this gene was analyzed by bisulfite sequencing and verified by DNA methylation-specific polymerase chain reaction. The results showed that the DNA methylation rate of CpG islands in the CDC2L1 gene promoter region was 50.0% (12/24) in patient keloid tissues and 0% (0/24) in normal skin-tissues from healthy controls. Patient keloid tissues with (n = 12) DNA methylation of the CDC2L1 gene promoter showed higher growth rates than those without (n = 12). Samples from keloid tissues with DNA methylation of the CDC2L1 gene promoter region had dramatically lower levels of CDK11p58 protein than samples from keloid tissues without DNA methylation of the CDC2L1 gene promoter region or healthy normal skin-tissues. In the fibroblasts with DNA methylation of the CDC2L1 gene promoter region from keloid tissues treated with DNA methyl-transferase inhibitor 5 aza 2'-deoxycytidine (5-aza-dC) for 48h, CDK11p58 levels in the fibroblasts were significantly increased in a dose-dependent manner; the apoptotic rate of the fibroblasts was significantly higher in the treated group than in the non-treated group. This study revealed that DNA methylation exists in the CDC2L1 gene promoter region in keloid tissue fibroblasts. DNA methylation of the CDC2L1 gene promoter region dramatically inhibits the expression of CDK11p58 protein in keloid tissues. A specific demethylation drug, 5-aza-dC, suppressed DNA methylation of the promoter region, which increased the expression of CDK11p58. The elevated expression of CDK11p58 resulted in increased fibroblast apoptosis, thus restraining the development of keloids.

Green Tea Extract and (−)-Epigallocatechin-3-Gallate Inhibit Mast Cell-Stimulated Type I Collagen Expression in Keloid Fibroblasts via Blocking PI-3K/Akt Signaling Pathways

Reticulocalbin 1 (RCN1) was reported to be upregulated in keloid, but its molecular mechanism remains unclear. The aim of this study is to investigate the role of RCN1 in keloid. The expression of RCN1 was detected in keloid tissues. Keloid fibroblasts were transfected with RCN1 overexpression vector. Cell viability, collagen production, apoptosis, and cell invasion were measured. Then, the m6A modification level of RCN1 mRNA was detected by methylated RNA immunoprecipitation (MeRIP), and the effect of overexpression of ALKB homolog5 (ALKBH5) on the m6A modification level of RCN1 mRNA was evaluated. Subsequently, the relationship between RCN1 and XBP1 was verified by co-immunoprecipitation (Co-IP) assay. pcDNA-RCN1 and XBP1 shRNA were transfected into keloid fibroblasts to for reversal experiments, and changes in the endoplasmic reticulum (ER) structure of keloid fibroblasts were observed by transmission electron microscopy (TEM). Finally, we established a mouse keloid model and injected mice with the RCN1 shRNA lentiviral vectors to monitor the keloid formation in mice. RCN1 was highly expressed in keloid tissues and keloid fibroblasts. Overexpression of RCN1 significantly increased keloid fibroblast viability, collagen production, and invasion, but inhibited cell apoptosis. ALKBH5 upregulated RCN1 expression by reducing m6A-YTHDF2-mediated degradation of RCN1 mRNA, and RCN1 knockdown reversed the promoting effect of ALKBH5 overexpression on cell viability collagen production and invasion, and the inhibitory effect of ALKBH5 overexpression on apoptosis in keloid fibroblasts. Moreover, overexpression of RCN1 significantly upregulated the protein levels of XBP1, GRP78, and IRE1α, and promoted ER stress in keloid fibroblasts, but this change was eliminated by sh-XBP1 intervention. Invivo experiments showed that knockdown of RCN1 significantly inhibited keloid formation by alleviating cell apoptosis and ER stress in mice. Our data revealed that RCN1 was upregulated by ALKBH5 to promote keloid formation by activating IRE1α-XBP1-mediated ER stress, RCN1 may be a potential biomarker for treatment of keloid.

Keloid, a fibroproliferative disorder, significantly impacts patients' quality of life, yet effective therapies remain elusive. This study explored the role of silent information regulator 6 (SIRT6) in modulating the proliferation, invasion, and collagen synthesis of keloid fibroblasts. Keloid and normal skin specimens were collected, and fibroblasts were isolated from the keloid tissue. SIRT6 recombinant adenovirus (Ad) was constructed to infect keloid fibroblasts to overexpress SIRT6. This study entails three groups: Control group, adenovirus-Negative Control (Ad-NC) group, and Ad-SIRT6 group. SIRT6 protein and mRNA levels were measured via Western blotting and Quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Cell viability was determined using 5-ethynyl-2'-deoxyuridine (EdU) assay. Flow cytometry was exploited to measure cell apoptosis. To investigate cell migration, wound healing assay and Transwell assay were employed. Western blotting was also utilized to study the expression levels of apoptotic proteins, collagen deposition-related proteins, and Mitogen-Activated Protein Kinases (MAPK)/extracellular regulated protein kinases (ERK) pathway-related proteins. Compared to the control and Ad-NC groups, the Ad-SIRT6 group exhibited significantly elevated SIRT6 level; diminished cell proliferation, migration and invasion; reduced protein levels of α-smooth muscle actin (α-SMA), collagen I, collagen III, phospho SMAD Family Member 3 (p-Smad3), transforming growth factor-β 1 (TGF-β1), and MAPK/ERK pathway proteins (phospho extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), phospho MAP kinase-ERK kinase (p-MEK) and phospho-c-Raf (p-c-Raf)). Treatment with epidermal growth factor (EGF), an MAPK/ERK pathway agonists, reversed the inhibitory effect of SIRT6 on cell activity and inhibited apoptosis in keloid fibroblasts. SIRT6 overexpression in keloid fibroblasts attenuates proliferation, invasion, and collagen synthesis, while fostering apoptosis, likely through the suppression of MAPK/ERK pathway activity. This suggests a potential therapeutic target for keloid treatment.

This study was intended to clarify the potential of applying the long-chain noncoding RNA H19/miR-29a axis in keloid treatment by elucidatingits correlation with the activity of fibroblasts. In this study, 80 keloid tissues, 63 normal fibrous tissues, and 91 normal skin tissues were collected in advance, and concurrently, fibroblasts separated from the tissues were cultured. Besides this, the si-H19, pcDNA3.1-H19, miR-29a mimic, and miR-29a inhibitor were transfected to keloid fibroblasts, whose proliferation, apoptosis, and metastasis were appraised by employing the colony formation assay, flow cytometry, and transwell assay. In addition, the luciferase reporter gene assay was carried out to determine whether targeted regulation was present between H19 and miR-29a, as well as between miR-29a and COL1A1. The study results demonstrated that keloid tissues and fibroblasts exhibited observably upregulated H19 expression and downregulated miR-29a expression, relative to normal skin tissues and fibroblasts (P < .05). Also observed was a negative correlation between H19 expression and miR-29a expression among the gathered keloid tissues (rs = -.267, P = .017). Furthermore, in vitro transfection of pcDNA3.1-H19 or miR-29a inhibitor could intensify viability, proliferation, migration, and invasion of the fibroblasts (P < .05), while silencing of H19 and overexpression of miR-29a hindered both metastasis and multiplication of the fibroblasts significantly (P < .05). In addition, H19 was capable of altering miR-29a expression within fibroblasts by directly sponging it, and overexpression of COL1A1 could deter the impact of miR-29a on viability, proliferation, migration, and invasion of fibroblasts (P < .05). In conclusion, H19 might facilitate proliferation and metastasis of fibroblasts by modifying downstream miR-29a and COL1A1, which was expected to allowfor development of keloid-targeted treatments.

The stress response following burns may be a crucial factor in keloid formation, yet the underlying pathological mechanisms remain to be elucidated. This study initially investigated how heat shock factor 1 (HSF1) and heat shock proteins (HSPs) within the heat shock pathway influence keloid fibrosis, providing insights into the role of the heat shock response in keloid development. This study aims to further elucidate the role of the heat shock pathway in keloid fibrosis and investigate the specific function of HSF1 within this pathway. This study focused on human keloid fibroblasts, examining the expression and regulatory role of HSF1 on HSPs under heat stress using immunohistochemistry, RNA interference, real-time fluorescent PCR, and Western blotting techniques. HSF1 was overexpressed in keloid fibroblasts and tissues compared to normal skin, and heat stress could further enhance HSF1 expression in both keloid tissues and fibroblasts. Functional inhibition of HSF1 significantly affected the expression of downstream HSPs in keloid fibroblasts, ultimately leading to the inhibition of keloid fibrosis. The heat shock pathway plays a crucial role in keloid fibrosis, with HSF1 as a key regulator influencing the expression of HSPs. Heat stress treatment of keloid fibroblasts offers an approach for investigating keloid formation.