[Screening, functional analysis and clinical validation of differentially expressed genes in diabetic foot ulcers].

Objective: To screen the differentially expressed genes (DEGs) in diabetic foot ulcers (DFUs), and to perform functional analysis and clinical validation of them, intending to lay a theoretical foundation for epigenetic therapy of chronic refractory wounds. Methods: An observational study was conducted. The gene expression profile dataset GSE80178 of DFU patients in Gene Expression Omnibus (GEO) was selected, and the DEG between three normal skin tissue samples and six DFU tissue samples in the dataset was analyzed and screened using the GEO2R tool. For the screened DEG, ClusterProfiler, org.Hs.eg.db, GOplot, and ggplot2 in the R language packages were used for Gene Ontology (GO) enrichment analysis of biological processes, molecular functions, and cellular components, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, respectively. Protein-protein interaction (PPI) analysis was performed using STRING database to screen key genes in the DEG, and GO enrichment analysis of key genes was performed using Cytohubba plug-in in Cytoscape 3.9.1 software. DFU tissue and normal skin tissue discarded after surgery were collected respectively from 15 DFU patients (7 males and 8 females, aged 55-87 years) and 15 acute wound patients (6 males and 9 females, aged 8-52 years) who were admitted to Xiang'an Hospital of Xiamen University from September 2018 to March 2021. The mRNA and protein expressions of small proline-rich repeat protein 1A (SPRR1A) and late cornified envelope protein 3C (LCE3C) were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Data were statistically analyzed with independent sample t test. Results: Compared with normal skin tissue, 492 statistically differentially expressed DEGs were screened from DFU tissue of DFU patients (corrected P<0.05 or corrected P<0.01), including 363 up-regulated DEGs and 129 down-regulated DEGs. GO terminology analysis showed that DEGs were significantly enriched in the aspects of skin development, keratinocyte (KC) differentiation, keratinization, epidermal development, and epidermal cell differentiation, etc. (corrected P values all <0.01). KEGG pathway analysis showed that DEGs were significantly enriched in the aspects of tumor-associated microRNA, Ras related protein 1 signaling pathway, and pluripotent stem cell regulatory signaling pathway, etc. (corrected P values all <0.01). PPI analysis showed that endophial protein, SPRR1A, SPRR1B, SPRR2B, SPRR2E, SPRR2F, LCE3C, LCE3E, keratin 16 (all down-regulated DEGs), and filoprotein (up-regulated DEG) were key genes of DEGs screened from DFU tissue of DFU patients, which were significantly enriched in GO terms of keratinization, KC differentiation, epidermal cell differentiation, skin development, epidermis development, and peptide cross-linking, etc. (corrected P values all <0.01). The mRNA expressions of SPRR1A and LCE3C in DFU tissue of DFU patients were 0.588±0.082 and 0.659±0.098, respectively, and the protein expressions were 0.22±0.05 and 0.24±0.04, respectively, which were significantly lower than 1.069±0.025 and 1.053±0.044 (with t values of 20.91 and 13.66, respectively, P values all <0.01) and 0.38±0.04 and 0.45±0.05 (with t values of 9.69 and 12.46, respectively, P values all <0.01) in normal skin tissue of acute wound patients. Conclusions: Compared with normal skin tissue, there is DEG profile in DFU tissue of DFU patients, with DEGs being significantly enriched in the aspects of KC differentiation and keratin function. Key DEGs are related to the biological function of KC, and their low expressions in DFU tissue of DFU patients may impede ulcer healing.

Cell Adhesion Molecules Affected by Ionizing Radiation and Estrogen in an Experimental Breast Cancer Model.

Cancer develops in a multi-step process where environmental carcinogenic exposure is a primary etiological component, and where cell-cell communication governs the biological activities of tissues. Identifying the molecular genes that regulate this process is essential to targeting metastatic breast cancer. Ionizing radiation can modify and damage DNA, RNA, and cell membrane components such as lipids and proteins by direct ionization. Comparing differential gene expression can help to determine the effect of radiation and estrogens on cell adhesion. An in vitro experimental breast cancer model was developed by exposure of the immortalized human breast epithelial cell line MCF-10F to low doses of high linear energy transfer α particle radiation and subsequent growth in the presence of 17ß-estradiol. The MCF-10F cell line was analyzed in different stages of transformation that showed gradual phenotypic changes including altered morphology, increase in cell proliferation relative to the control, anchorage-independent growth, and invasive capability before becoming tumorigenic in nude mice. This model was used to determine genes associated with cell adhesion and communication such as E-cadherin, the desmocollin 3, the gap junction protein alpha 1, the Integrin alpha 6, the Integrin beta 6, the Keratin 14, Keratin 16, Keratin 17, Keratin 6B, and the laminin beta 3. Results indicated that most genes had greater expression in the tumorigenic cell line Tumor2 derived from the athymic animal than the Alpha3, a non-tumorigenic cell line exposed only to radiation, indicating that altered expression levels of adhesion molecules depended on estrogen. There is a significant need for experimental model systems that facilitate the study of cell plasticity to assess the importance of estrogens in modulating the biology of cancer cells.

A unique skin phenotype resulting from a large heterozygous deletion spanning six keratin genes.

The phenotypic spectrum of genodermatoses is continuously expanding. Three siblings were referred because of a highly unusual phenotype comprising alopecia, dystrophic nails, palmoplantar keratoderma and trauma-induced skin blistering. Whole-exome sequencing analysis identified a heterozygous large genomic alteration of around 116 0000 bp resulting in the deletion of the KRT9, KRT14, KRT15, KRT16 and KRT19 genes, as well as part of KRT17. This genomic change leads to the generation of a truncated keratin 17 (KRT17) protein encoded by the first three exons of the gene and part of intron 3. The three patients were found to carry the heterozygous genomic deletion while their healthy parents did not, indicative of germline mosaicism. The genomic alteration was found to result in reduced KRT17 expression in patient skin. More importantly, the abnormal truncated KRT17 was found to exert a deleterious effect on keratinocyte cytoskeleton formation, leading to keratin aggregation. Coexpression of wildtype and truncated KRT17 proteins also caused keratin aggregation, demonstrating that the deletion exerts a dominant negative effect. In conclusion, we are reporting on a novel clinical phenotype that was found to result from germline mosaicism for a large genomic deletion spanning six keratin genes, thus expanding the spectrum of clinical manifestations associated with keratin disorders. What is already known about this topic? Various conditions known as keratinopathies have been shown over recent years to be associated with dominant or recessive variants in several individual keratin genes. What does this study add? We report three patients presenting with a unique clinical phenotype that was found to result from germline mosaicism for a large genomic deletion spanning six keratin genes. The genomic variant is predicted to result in a truncated form of keratin 17, which was found in an in vitro assay to disrupt keratinocyte cell cytoskeleton formation.

Activation of the C3a anaphylatoxin receptor inhibits keratinocyte proliferation by regulating keratin 6, keratin 16, and keratin 17 in psoriasis.

Emerging evidence suggests that signaling through the C3a anaphylatoxin receptor (C3aR) protects against various inflammation-related diseases. However, the role of C3aR in psoriasis remains unknown. The purpose of this study was to investigate the possible protective role of C3aR in psoriasis and to explore the underlying molecular mechanisms. We initially found that the psoriatic epidermis exhibited significantly decreased C3aR expression. C3aR showed protective roles in mouse models of imiquimod (IMQ)- and interleukin-23-induced psoriasis. Furthermore, increased epidermal thickness and keratin 6 (K6), K16, and K17 expression occurred in the ears and backs of C3aR-/- mice. Pharmacological treatment with a C3aR agonist ameliorated IMQ-induced psoriasiform lesions in mice and decreased the expression of K6, K16, and K17. Additionally, the signal transducer and activator of transcription 3 (STAT3) pathway participated in the protective function of C3aR. More importantly, the expression levels of K6, K16, and K17 in keratinocytes were all restored in HaCaT cells transfected with a C3aR-overexpression plasmid after treating them with colivelin (a STAT3 activator). Our findings demonstrate that C3aR protects against the development of psoriasis and suggest that C3aR confers protection by negatively regulating K6, K16, and K17 expression in a STAT3-dependent manner, thus inhibiting keratinocyte proliferation and helping reverse the pathogenesis of psoriasis.

BarH-like homeobox 2 represses the transcription of keratin 16 and affects Ras signaling pathway to suppress nasopharyngeal carcinoma progression.

Nasopharyngeal carcinoma (NPC) refers to a malignancy initiating from the superior mucosal epithelium of the nasopharynx. Optimal therapies for NPC are still needed. In this investigation, we attempted to explore whether BarH-like homeobox 2 (BARX2), a well-known tumor suppressor, had anti-cancer properties on NPC, and the possible mechanisms. After searching for NPC-related databases, we determined BARX2 as one of the core genes in NPC. The results of RT-qPCR and immunohistochemistry or Western blot demonstrated that BARX2 was reduced in NPC patients and cells. Ectopic expression of BARX2 reverted the malignant phenotype of NPC cells. Mechanistically, BARX2 bound to the keratin 16 (KRT16) promoter to downregulate its expression. In addition, BARX2 was found to reduce the phosphorylation levels of MEK and ERK. Further KRT16 upregulation in cells overexpressing BARX2 promoted malignant aggressiveness of C666-1 and HNE3 cells and activated the Ras signaling pathway. BARX2 inhibited the growth and metastasis of tumors and suppressed the Ras signaling pathway in vivo. In conclusion, our findings indicate that BARX2 reverts malignant phenotypes of NPC cells by downregulating KRT16 in a Ras-dependent fashion. BARX2 might act as a possible therapeutic regulator for NPC.

Genotype‒Structurotype‒Phenotype Correlations in Patients with Pachyonychia Congenita.

Pachyonychia congenita (PC) is a genetic disorder of keratin that presents with nail dystrophy, painful palmoplantar keratoderma, and other clinical manifestations. We investigated the genotype‒structurotype‒phenotype correlations seen with mutations in keratin genes (keratin [K]6A, K6B, K6C, K16, K17) and utilized protein structure modeling of high-frequency mutations to examine the functional importance of keratin structural domains in PC pathogenesis. Participants of the International PC Research Registry underwent genetic testing and completed a standardized survey on their symptoms. Our results support previous reports associating oral leukokeratosis with K6A mutations and cutaneous cysts, follicular hyperkeratosis, and natal teeth with K17 mutations. Painful keratoderma was prominent with K6A and K16 mutations. Nail involvement was most common in patients with K6A mutation and least common in those with K6C mutation. Across keratin subtypes, patients with coil 2B mutations had the greatest impairment in ambulation, and patients with coil 1A mutations reported more emotional issues. Molecular modeling demonstrated that hotspot missense mutations in PC largely disrupted hydrophobic interactions or surface charge. The former may destabilize keratin dimers/tetramers, whereas the latter likely interferes with higher-order keratin filament formation. Understanding the pathologic alterations in keratin structure improves our knowledge of how PC genotype correlates with clinical phenotype, advancing insight into disease pathogenesis and therapeutic development.

Evaluation of expression of Toll-Like Receptors 7 and 9, proliferation, and cytoskeletal biomarkers in plaque and guttate psoriasis: A pilot morphological study.

This pilot study was aimed at comparing TLR7/TLR9 expression, cytoskeletal arrangement, and cell proliferation by indirect immunofluorescence in parallel lesional and non lesional skin samples of guttate psoriasis (PG) and psoriasis vulgaris (PV) in five male patients for each group (n=10). TLR7 expression was detected throughout all the epidermal compartment in PV samples, while in PG skin was restricted to the granular layer. TLR9 was present in the granular layer of non lesional skin and in the suprabasal layers of PV/PG lesional skin. Cell proliferation was localized in all the epidermal layers in lesional PG and PV, consistently with the immunopositivity for the "psoriatic keratin" K16. In the suprabasal layers of lesional PG and PV skin, a similar K17 expression was detected and K10 exhibited a patchy distribution. The present results suggest that TLR7 expression can be considered an intrinsic and differential histomorphological feature of PV.

LncRNA NORAD engages in psoriasis by binding to miR-26a to regulate keratinocyte proliferation.

BACKGROUND: Psoriasis is a chronic, inflammatory skin disease. It was reported that lncRNA Non-coding RNA-activated by DNA damage (NORAD) has potential regulatory effects on skin diseases. Our previous studies found that lncRNA NORAD was highly expressed and its potential target miR-26a was down-regulated in psoriasis model mice. Here, we aimed to investigate the role of NORAD in the development of psoriasis. METHODS: IL-22/LPS (interleukin-22/lipopolysaccharide)-stimulated HaCaT (human immortalized keratinocytes) cell model and imiquimod-induced mouse model were established. Keratin 6 (K6), Keratin 16 (K16), Keratin 17 (K17), and Cell division cycle 6 (CDC6) levels were detected by western blot. Cell activity was detected by CCK-8, MTT, and EdU assays. Quantitative real-time PCR was performed to examine the levels of NORAD, miR-26a, CDC6, K6, K16, and K17. Haematoxylin-eosin staining was applied to observe the degree of skin thickening and hyperplasia. Fluorescence in situ hybridization detects the location of NORAD. RNA immunoprecipitation, RNA pull-down, and Luciferase test were performed to detect the interaction between NORAD and miR-26a. RESULTS: In IL-22/LPS-stimulated HaCaT cells, NORAD, CDC6, and keratinocyte proliferation-related proteins (K6, K16, and K17) were up-regulated and miR-26a was down-regulated. Cell survival and proliferation were also increased. However, the results were reversed after interference with NORAD. Also, in vitro experiments revealed that NORAD negatively regulated miR-26a. In IL-22/LPS-stimulated HaCaT cells and skin of imiquimod-induced mice, we found that lower NORAD resulted in an increase of miR-26a and a decrease of CDC6, further decreased levels of keratinocyte proliferation-related proteins (K6, K16, and K17).

Identification of clinically useful predictive genetic variants in pachyonychia congenita.

BACKGROUND: Pachyonychia congenita (PC) refers to a group of autosomal dominant disorders caused by mutations in five keratin genes (KRT16,KRT6A,KRT17,KRT6B or KRT6C). Current disease classification is based on the gene harbouring disease-causing variants. AIMS: We harnessed the International Pachyonychia Congenita Research Registry (IPCRR) containing both clinical and molecular data on patients with PC worldwide, to identify genetic variants predicting disease severity. METHODS: We ascertained 815 individuals harbouring keratin mutations registered in the IPCRR. We looked for statistically significant associations between genetic variants and clinical manifestations in a subgroup of patients carrying mutations found in at least 10% of the cohort. Data were analysed using χ2 and Kruskal-Wallis tests. RESULTS: We identified five mutations occurring in at least 10% of the patients registered in the IPCRR. The KRT16 p.L132P mutation was significantly associated with younger age of onset, presence of palmar keratoderma oral leucokeratosis and a higher number of involved nails. By contrast, the KRT16 p.N125S and p.R127C mutations resulted in a milder phenotype featuring a decreased number of involved nails and older age of onset. Patients carrying the p.N125S mutation were less likely to develop palmar keratoderma while p.R127C was associated with an older age of palmoplantar keratoderma onset. Moreover, the KRT17 p.L99P mutation resulted in an increased number of involved fingernails and patients demonstrating 20-nail dystrophy, while the opposite findings were observed with KRT17 p.N92S mutation. CONCLUSIONS: We have identified novel and clinically useful genetic predictive variants in the largest cohort of patients with PC described to date.

Development and characterization of a human Th17-driven ex vivo skin inflammation model.

Skin models mimicking features of psoriasis-related inflammation are needed to support the development of new drugs in dermatology. Reconstructed skin models lack tissue complexity, including a fully competent skin barrier, and presence and/or diversity of immune cells. Here, we describe InflammaSkin®, a novel human Th17-driven ex vivo skin inflammation model. In this model, skin-resident T cells are in situ activated by intradermal injection of anti-CD3 and anti-CD28 antibodies and Th17 cell polarization is sustained by culture in a chemically defined medium supplemented with IL-1ß, IL-23 and TGF-ß for seven days. The acquired Th17 signature is demonstrated by the sustained secretion of IL-17A, IL-17AF, IL-17F, IL-22, IFN-γ, and to some degree IL-15 and TNF-α observed in the activated ex vivo skin inflammation model compared with the non-activated skin model control. Furthermore, expression of S100A7 and Keratin-16 by keratinocytes and loss of epidermal structure integrity occur subsequently to in situ Th17cell activation, demonstrating cellular crosstalk between Th17 cells and keratinocytes. Finally, we demonstrate the use of this model to investigate the modulation of the IL-23/IL-17 immune axis by topically applied anti-inflammatory compounds. Taken together, we show that by in situ activation of skin-resident Th17 cells, the InflammaSkin® model reproduces aspects of inflammatory responses observed in psoriatic lesions and could be used as a translational tool to assess efficacy of test compounds.

A role for keratins in supporting mitochondrial organization and function in skin keratinocytes.

Mitochondria fulfill essential roles in ATP production, metabolic regulation, calcium signaling, generation of reactive oxygen species (ROS), and additional determinants of cellular health. Recent studies have highlighted a role for mitochondria during cell differentiation, including in skin epidermis. The observation of oxidative stress in keratinocytes from Krt16 null mouse skin, a model for pachyonychia congenita (PC)-associated palmoplantar keratoderma, prompted us to examine the role of Keratin (K) 16 protein and its partner K6 in regulating the structure and function of mitochondria. Electron microscopy revealed major anomalies in mitochondrial ultrastructure in late stage, E18.5, Krt6a/Krt6b null embryonic mouse skin. Follow-up studies utilizing biochemical, metabolic, and live imaging readouts showed that, relative to controls, skin keratinocytes null for Krt6a/Krt6b or Krt16 exhibit elevated ROS, reduced mitochondrial respiration, intracellular distribution differences, and altered movement of mitochondria within the cell. These findings highlight a novel role for K6 and K16 in regulating mitochondrial morphology, dynamics, and function and shed new light on the causes of oxidative stress observed in PC and related keratin-based skin disorders.

Histopathology and selective biomarker expression in human meibomian glands.

BACKGROUND/AIMS: Meibomian gland dysfunction (MGD) is the most common form of evaporative dry eye disease, but its pathogenesis is poorly understood. This study examined the histopathological features of meibomian gland (MG) tissue from cadaver donors to identify potential pathogenic processes that underlie MGD in humans. METHODS: Histological analyses was performed on the MGs in the tarsal plates dissected from four cadaver donors, two young and two old adults, including a 36-year-old female (36F) and three males aged 30, 63 and 64 years (30M, 63M and 64M). RESULTS: The MGs of 36F displayed normal anatomy and structure, whereas the MGs of 30M showed severe ductal obstruction with mild distortion. The obstruction was caused by increased cytokeratin levels in association with hyperproliferation, but not hyperkeratinisation. In two older males, moderate to severe MG atrophy was noted. Cell proliferation was significantly reduced in the MG acini of the two older donors as measured by Ki67 labelling index (6.0%±3.4% and 7.9%±2.8% in 63M and 64M, respectively) when compared with that of the two younger donors (23.2%±5.5% and 16.9%±4.8% in 30M and 36F, respectively) (p<0.001). The expression patterns of meibocyte differentiation biomarkers were similar in the older and younger donors. CONCLUSION: Our histopathological study, based on a small sample size, suggests potentially distinct pathogenic mechanisms in MGD. In the young male adult, hyperproliferation and aberrant differentiation of the central ductal epithelia may lead to the obstruction by overproduced cytokeratins. In contrast, in older adults, decreased cell proliferation in acinar basal epithelia could be a contributing factor leading to MG glandular atrophy.