Hypoxia macrophage-derived exosomal miR-26b-5p targeting PTEN promotes the development of keloids.

Background: Hypoxia is the typical characteristic of keloids. The development of keloids is closely related to the abnormal phenotypic transition of macrophages. However, the role of exosomal microRNAs (miRNAs) derived from hypoxic macrophages in keloids remains unclear. This study aimed to explore the role of hypoxic macrophage-derived exosomes (HMDE) in the occurrence and development of keloids and identify the critical miRNA. Methods: The expression of CD206+ M2 macrophage in keloids and normal skin tissues was examined through immunofluorescence. The polarization of macrophages under a hypoxia environment was detected through flow cytometry. The internalization of macrophage-derived exosomes in human keloid fibroblasts (HKFs) was detected using a confocal microscope. miRNA sequencing was used to explore the differentially expressed miRNAs in exosomes derived from the normoxic and hypoxic macrophage. Subsequently, the dual-luciferase reporter assay verified that phosphatase and tension homolog (PTEN) was miR-26b-5p's target. The biological function of macrophage-derived exosomes, miR-26b-5p and PTEN were detected using the CCK-8, wound-healing and Transwell assays. Western blot assay was used to confirm the miR-26b-5p's underlying mechanisms and PTEN-PI3K/AKT pathway. Results: We demonstrated that M2-type macrophages were enriched in keloids and that hypoxia treatment could polarize macrophages toward M2-type. Compared with normoxic macrophages-derived exosomes (NMDE), HMDE promote the proliferation, migration and invasion of HKFs. A total of 38 differential miRNAs (18 upregulated and 20 downregulated) were found between the NMDE and HMDE. miR-26b-5p was enriched in HMDE, which could be transmitted to HKFs. According to the results of the functional assay, exosomal miR-26b-5p produced by macrophages facilitated HKFs' migration, invasion and proliferation via the PTEN-PI3K/AKT pathway. Conclusions: The highly expressed miR-26b-5p in HMDE promotes the development of keloids via the PTEN-PI3K/AKT pathway.

SUMOylation mediates the disassembly of the Smad4 nuclear export complex via RanGAP1 in KELOIDS.

Sentrin/small ubiquitin-like modifier (SUMO) has emerged as a powerful mediator regulating biological processes and participating in pathophysiological processes that cause human diseases, such as cancer, myocardial fibrosis and neurological disorders. Sumoylation has been shown to play a positive regulatory role in keloids. However, the sumoylation mechanism in keloids remains understudied. We proposed that sumoylation regulates keloids via a complex. RanGAP1 acted as a synergistic, functional partner of SUMOs in keloids. Nuclear accumulation of Smad4, a TGF-ß/Smad pathway member, was associated with RanGAP1 after SUMO1 inhibition. RanGAP1*SUMO1 mediated the nuclear accumulation of Smad4 due to its impact on nuclear export and reduction in the dissociation of Smad4 and CRM1. We clarified a novel mechanism of positive regulation of sumoylation in keloids and demonstrated the function of sumoylation in Smad4 nuclear export. The NPC-associated RanGAP1*SUMO1 complex functions as a disassembly machine for the export receptor CRM1 and Smad4. Our research provides new perspectives for the mechanisms of keloids and nucleocytoplasmic transport.

Comprehensive Analysis of Circular RNA Expression in ceRNA Networks and Identification of the Effects of hsa_circ_0006867 in Keloid Dermal Fibroblasts.

Circular RNAs (circRNAs) play a crucial role in the pathogenesis of various fibrotic diseases, but the potential biological function and expression profile of circRNAs in keloids remain unknown. Herein, microarray technology was applied to detect circRNA expression in four patient-derived keloid dermal fibroblasts (KDFs) and normal dermal fibroblasts (NDFs). A total of 327 differentially expressed (DE) circRNAs (fold change > 1.5, p < 0.05) were identified with 195 upregulated and 132 downregulated circRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that the upregulated circRNAs were mainly enriched in the cytoskeleton and tight junctions, while the downregulated circRNAs were related to morphogenesis of the epithelium and axonal guidance. To explore the function of DE circRNAs, a circRNA-miRNA-mRNA network, including five circRNAs, nine miRNAs, and 235 correlated mRNAs, was constructed using bioinformatics analyses. The expression of five DE circRNAs was validated by qRT-PCR in 18 pairs of KDFs and NDFs, and hsa_circ_0006867 showed promising regulatory function in keloids in vitro. Silencing hsa_circ_00006867 suppressed the proliferation, migration, and invasion of keloid fibroblasts. RNA-binding protein immunoprecipitation (RIP) assays indicated that hsa_circ_00006867 may serve as a platform for miRNA binding to Argonaute (AGO) 2. In addition, hsa-miR-29a-5p may be a potential target miRNA of hsa_circ_00006867. Taken together, our research provided multiple novel clues to understand the pathophysiologic mechanism of keloids and identified hsa_circ_0006867 as a biomarker of keloids.

Metformin Inhibits Epithelial-to-Mesenchymal Transition of Keloid Fibroblasts via the HIF-1α/PKM2 Signaling Pathway.

Background: Epithelial-to-mesenchymal transition (EMT) is a process whereby epithelial cells lose cell-cell contacts and acquire expression of mesenchymal components and manifest a migratory phenotype. Recent studies indicated that EMT is involved in the development of keloids. Therefore, this study aims to investigate the mechanisms of the effects of metformin in hypoxia-induced EMT in keloid fibroblasts (KFs). Methods: KFs were cultured in a hypoxia incubator to induce EMT and were treated with or without metformin. Cell viability was evaluated by a cell counting kit 8 (CCK-8), and cell migration was measured by the transwell assay. The expression levels of HIF-1α, E-cadherin, vimentin, phosphorylated p70s6k (p-p70s6k) and pyruvate kinase M2 (PKM2) were evaluated by western blotting. Results: Hypoxia promoted EMT in KFs. Metformin significantly inhibited the expression of HIF-1α and partially abolished hypoxia-induced EMT. PKM2 is involved in hypoxia-induced EMT of KFs and metformin decreased the expression of p-p70s6k and PKM2. Conclusions: Metformin abolishes hypoxia-induced EMT in KFs by inhibiting the HIF-1α/PKM2 signaling pathway. Our study provides a novel mechanistic insight into potential use of metformin for treatment of keloids.

Inactivating the ubiquitin ligase Parkin suppresses cell proliferation and induces apoptosis in human keloids.

Keloids are a common type of pathological skin healing, characterized by the destruction of the vascular network. Thus, keloids often exhibit anoxic conditions. Hypoxia-inducible factor-1α (HIF-1α) is a core factor that mediates hypoxia stress responses and allows the cells to adapt to low-oxygen conditions. In the current study, we identified that Parkin acted as an E3 ubiquitin ligase, contributing to the degradation of HIF-1α in keloid fibroblasts (KFs). Silencing of Parkin in KFs upregulated HIF-1α expression and prolonged its protein half-life. Furthermore, Parkin influenced transforming growth factor ß (TGF-ß)/Smad signaling by targeting HIF-1α. Under hypoxic conditions, silencing Parkin enhanced KF proliferation and inhibited apoptosis through the TGF-ß/Smad signaling pathway. Notably, metformin, an antidiabetic drug, could significantly induce Parkin expression and enhance the interaction between Parkin and HIF-1α. As a result, we revealed an important mechanism for Parkin in keloid development and suggested that targeting Parkin could be an alternative method for keloid treatment.

Universal DNA biosensing based on instantaneously electrostatic attraction between hexaammineruthenium (III) and DNA molecules.

Despite rapid progress in DNA biosensors by employing various materials as well as techniques, most of the reported sensors are based on specific recognition of a DNA fragment, however can not perform universal measurement of DNA molecules (i.e. genomic DNA). In this work, we proposed a novel DNA biosensing method based on instantaneously electrostatic attraction (IEA) between hexaammineruthenium (III) and DNA molecules. The current variation of freely diffused Ru(NH3)63+ caused by its quick and strong static interaction with phosphate backbones was employed as a universal probe to detect DNA molecules in solution, with no need for immobilization of capture probes on the electrode. After optimization, 30 µL of 300 µM Ru(NH3)63+ solution was added onto the gold electrode with a working electrode diameter of 2 mm, and a detection limit of 3.8 ng/µL was achieved, which is equivalent to NanoDrop™ One spectrometer, the commonly used instrument for DNA quantification. Using reusable and inexpensive gold electrode, the approach provided an easy-operated sequence-independent DNA detection method, and was proved to be able to detect genomic and plasmid DNA directly.