Role of interleukin-36γ induced by ultraviolet radiation in chronic actinic dermatitis.

BACKGROUND: Chronic actinic dermatitis (CAD) is an immune-mediated photodermatosis characterized by a high eosinophil count and total immunoglobulin E (IgE) in the peripheral blood of patients. At present, however, the reasons for their elevation remain unclear. OBJECTIVE: The current study aimed to detect changes in inflammatory cytokines in CAD and explore their role in this disease. METHODS: Enzyme-linked immunosorbent assay and Luminex assay were conducted to measure inflammatory factor levels. Immunohistochemical analysis and quantitative real-time polymerase chain reaction were performed to evaluate the expression levels of interleukin-36γ (IL-36γ), IL-8, chemokine (C-C motif) ligand 17 (CCL17), and CCL18. CCK8 kits were used to assess cell proliferation. Immunofluorescence was used to detect nuclear factor κB (NF-κB) p65 nuclear translocation. Western blot analysis was performed to detect the protein expression level of phosphorylated NF-κB (p-NF-κB) p65. Hematoxylin and eosin and Masson trichrome staining were applied to observe histological changes in a chronic photo-damaged mouse model. RESULTS: Eosinophils, total IgE, IL-36γ, IL-8, tumor necrosis factor α, CCL17, and CCL18 were elevated in CAD. Of note, IL-36γ promoted the proliferation of eosinophilic cells (EOL-1) and the production of IgE in peripheral blood mononuclear cells. IL-36γ also promoted the production of IL-8 and CCL18 in immortalized human keratinocytes (HaCaT cells), while ultraviolet radiation (UVR)-induced IL-36γ via activation of the NF-κB signaling pathway. CONCLUSIONS: IL-36γ was involved in the pathogenesis of CAD and UVR contributed to the production of IL-36γ, which may provide a novel therapeutic target for CAD.

LncRNA-WAKMAR2 regulates expression of CLDN1 to affect skin barrier through recruiting c-Fos.

BACKGROUND: Chronic actinic dermatitis (CAD) is an immune-mediated photo-allergic skin disease. In the clinic, the treatment of this disease is hampered by the lack of proper understanding of the skin barrier dysfunction mechanism. OBJECTIVE: To illuminate the mechanism of skin barrier dysfunction in CAD. METHODS: Transcriptome sequencing and protein profiling were used to detect skin barrier injury-related genes. RNA pull down, a promoter-reporter gene assay, and chromatin isolation by RNA purification-sequencing were used to elucidate the effect of WAKMAR2 in skin barrier functionality. RESULTS: Transcriptome sequencing from patient's tissues showed a significantly decreased expression of WAKMAR2. Down-regulation of WAKMAR2 destroyed the keratinocyte barrier. Moreover, WAKMAR2 can directly bind to the c-Fos protein. This novel long non-coding RNA (LncRNA)-protein complexes were targeted to the CLDN1 promotor. Overexpression of WAKMAR2 enhanced the promoter activity of CLDN1, while the addition of AP-1 inhibitor could reverse this phenomenon. Furthermore, our in vivo results suggested that expression of WAKMAR2 was required for the repair of skin damage in mice induced by ultraviolet irradiation. CONCLUSIONS: We identified a crucial LncRNA (WAKMAR2) for the protection of the skin barrier in vitro and in vivo. Mechanically, it can specifically interact with c-Fos protein for the regulation of CLDN1, a finding which could be applied for CAD treatment.

Up-regulation of hsa-miR-221-3p induced by UVB affects proliferation and apoptosis of keratinocytes via Bcl-xL/Bax pathway.

BACKGROUND: Chronic actinic dermatitis (CAD) is a photoallergic skin disease with abnormal hyperplasia. At present, the mechanism of abnormal proliferation is not clear. OBJECTIVE: To explore possible mechanism of CAD proliferative lesions. METHODS: Immunohistochemistry (IHC) assay and small RNA sequencing were carried out. Quantitative real-time PCR (qRT-PCR) analysis was performed to evaluate expression levels of hsa-miR-221-3p and FOS. The interaction between hsa-miR-221-3p and FOS was identified by dual-luciferase reporter assay. Expression of hsa-miR-221-3p also was detected by qRT-PCR after UVB irradiation. Influences of hsa-miR-221-3p and FOS on cell viability and apoptosis were assessed through a series of functional experiments and rescue experiments. Western blot analysis was used to detect protein expression of fos, Bax, Bcl-xL, and caspase-3. RESULTS: Patients with CAD had marked epidermal hyperplasia. The expression of hsa-miR-221-3p was up-regulated in CAD while FOS was significantly down-regulated. Dual-luciferase reporter assay confirmed that hsa-miR-221-3p targeted FOS 3'UTR. Hsa-miR-221-3p induced by UVB ranged from 0 to 30 mJ. Moreover, hsa-miR-221-3p overexpression or FOS knockdown promoted cell proliferation and reduced cell apoptosis. Western blot showed that hsa-miR-221-3p negatively regulated fos, which regulated Bcl-xL/Bax. Cell proliferation caused by hsa-miR-221-3p overexpression or FOS knockdown could be reversed by Bcl-xL inhibitor. CONCLUSION: Hsa-miR-221-3p induced by UVB targeted FOS 3'UTR, which played an important role in regulating proliferation and apoptosis of keratinocytes via Bcl-xL/Bax pathway; this may provide a new insight for CAD proliferative lesions.

Upregulation of hsa-miR-31-3p induced by ultraviolet affects keratinocytes permeability barrier by targeting CLDN1.

Chronic actinic dermatitis (CAD) is a photoallergic skin disease with complicated pathogenesis. However, skin barrier dysfunction may be involved according to clinical manifestation. To investigate the mechanism of CAD barrier dysfunction, noninvasive detection of skin barrier and small RNA sequencing were carried out. Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression levels of hsa-miR-31-3p and CLDN1. The correlation between hsa-miR-31-3p and CAD severity was explored. Further, dual-luciferase reporter assay was performed to identify the relationship between hsa-miR-31-3p and CLDN1. In addition, expression of hsa-miR-31-3p was detected after ultraviolet (UV) irradiation. Influences of hsa-miR-31-3p on primary human keratinocytes barrier were assessed by FITC-Dextran permeability assay. Moreover, western blot was used to detect the expression of claudin-1, filaggrin, loricrin and involucrin. Our results showed that transepidermal water loss (TEWL) significantly increased in CAD, while stratum corneum hydration (SCH) significantly decreased. The expression of hsa-miR-31-3p was up-regulated in CAD while CLDN1 was down-regulated. Hsa-miR-31-3p was correlated with TEWL, UV-MED (minimal erythema dose) and clinical severity scores of CAD (CSS-CAD). Dual-luciferase reporter assay confirmed that hsa-miR-31-3p targeted the 3'UTR region of CLDN1. Moreover, hsa-miR-31-3p was induced by UVB (0-30 mJ/cm2) and UVA (0-4 J/cm2). Furthermore, overexpression of hsa-miR-31-3p increased FITC-Dextran flux of primary human keratinocytes and reduced the expression of claudin-1, filaggrin, loricrin and involucrin. In conclusion, we demonstrated that hsa-miR-31-3p induced by UV was correlated with CAD severity, which played an important role in regulating keratinocytes permeability barrier through targeting CLDN1.

[Effect of turmeric volatile oil on proliferation and apoptosis of human skin SCC A431 cells].

To investigate the effect of turmeric volatile oil (TVO) on the apoptosis and proliferation of human skin SCC A431 cells, A431 cells were incubated with different concentrations (5-80 mg•L⁻¹) of TVO in vitro．The proliferation and cell cycle were assessed by CCK8 assay. The change of morphology was observed with inverted microscope. Apoptosis was evaluated with AO/EB double staining and flow cytometry (FCM); cell cycle was analyzed with FCM ．Western blot method was used to detect caspase-3 and caspase-9 protein expression. Results indicated that TVO has significant inhibitory effects on the growth of A431 cells in a dose dependent relationship, the difference between groups has statistically significant (P<0.05). TVO group compared with control group, concentrations in cells shrivel and broken phenomenon, cell apoptosis rate increased, and a dose dependent and increased the expression of caspase-3 and caspase-9. The experiment results suggested that TVO could restrain skin squamous carcinoma A431 cells proliferation, and induce its apoptosis. The mechanism may be related to increase the expression of caspase-3 and caspase-9.

Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most frequent of the keratinocyte-derived malignancies with actinic keratosis (AK) as a precancerous lesion. To comprehensively delineate the underlying mechanisms for the whole progression from normal skin to AK to invasive cSCC, we performed single-cell RNA sequencing (scRNA-seq) to acquire the transcriptomes of 138,982 cells from 13 samples of six patients including AK, squamous cell carcinoma in situ (SCCIS), cSCC, and their matched normal tissues, covering comprehensive clinical courses of cSCC. We identified diverse cell types, including important subtypes with different gene expression profiles and functions in major keratinocytes. In SCCIS, we discovered the malignant subtypes of basal cells with differential proliferative and migration potential. Differentially expressed genes (DEGs) analysis screened out multiple key driver genes including transcription factors along AK to cSCC progression. Immunohistochemistry (IHC)/immunofluorescence (IF) experiments and single-cell ATAC sequencing (scATAC-seq) data verified the expression changes of these genes. The functional experiments confirmed the important roles of these genes in regulating cell proliferation, apoptosis, migration, and invasion in cSCC tumor. Furthermore, we comprehensively described the tumor microenvironment (TME) landscape and potential keratinocyte-TME crosstalk in cSCC providing theoretical basis for immunotherapy. Together, our findings provide a valuable resource for deciphering the progression from AK to cSCC and identifying potential targets for anticancer treatment of cSCC.

Cul1 promotes melanoma cell proliferation by promoting DEPTOR degradation and enhancing cap-dependent translation.

Cullin1 (Cul1) serves as a rigid scaffold in the SCF (Skp1/Cullin/Rbx1/F-box protein) E3 ubiquitin ligase complex and has been found to be overexpressed in melanoma and to enhance melanoma cell proliferation by promoting G1-S phase transition. However, the underlying mechanisms involved in the regulation of melanoma cell proliferation by Cul1 remain poorly understood. In the present study, we found that Cul1 promoted mTORC1 activity and cap-dependent translation by enhancing the ubiquitination and degradation of DEPTOR. We further showed that suppression of the eIF4F complex assembly profoundly inhibited the promoting effect of Cul1 on melanoma cell proliferation, while enhancement of the eIF4F complex activity reversed the inhibitory effect of Cul1 depletion on melanoma cell proliferation, indicating that Cul1 contributes to melanoma cell proliferation by activating cap­dependent translation. These data elucidate the role of Cul1 in cap-dependent translation and improves our understanding of the underlying mechanisms involved in the regulation of melanoma cell proliferation by Cul1.