早期蕈样肉芽肿微小RNA表达谱的研究

目的：筛选与早期蕈样肉芽肿（MF）相关的微小RNA（miRNA ）。方法用高通量miRNA PCR芯片检测6例早期MF与6例湿疹和扁平苔藓皮损中miRNA的表达差异。针对差异表达的miRNA，进行13例早期MF、13例湿疹和扁平苔藓皮损组织及Myla细胞株的实时荧光定量PCR（RT?qPCR）验证。结果芯片结果示，相对于对照组，早期MF hsa?miR?378a?5p、hsa?miR?107、hsa?miR?302c?3p显著高表达，差异有统计学意义（P<0.05）。皮损组织的RT?qPCR验证结果与芯片结果一致。与正常人外周血T淋巴细胞相比，Myla细胞株中hsa?miR?378a?5p、hsa?miR?107显著上调，与芯片结果一致；未见hsa?miR?302c?3p的差异性表达。结论与炎症性皮肤病相比，早期MF存在差异表达的miRNA表达谱。     

Laser capture microdissection followed by next‐generation sequencing identifies disease‐related microRNAs in psoriatic skin that reflect systemic microRNA changes in psoriasis

Psoriasis is a systemic disease with cutaneous manifestations. MicroRNAs (miRNAs) are small non‐coding RNA molecules that are differentially expressed in psoriatic skin; however, only few cell‐ and region‐specific miRNAs have been identified in psoriatic lesions. We used laser capture microdissection (LCM) and next‐generation sequencing (NGS) to study the specific miRNA expression profiles in the epidermis (Epi) and dermal inflammatory infiltrates (RD) of psoriatic skin (N = 6). We identified 24 deregulated miRNAs in the Epi and 37 deregulated miRNAs in the RD of psoriatic plaque compared with normal psoriatic skin (FCH > 2, FDR < 0.05). Interestingly, 9 of the 37 miRNAs in RD, including miR‐193b and miR‐223, were recently described as deregulated in circulating peripheral blood mononuclear cells (PBMCs) from patients with psoriasis. Using flow cytometry and qRT‐PCR, we found that miR‐193b and miR‐223 were expressed in Th17 cells. In conclusion, we demonstrate that LCM combined with NGS provides a robust approach to explore the global miRNA expression in the epidermal and dermal compartments of psoriatic skin. Furthermore, our results indicate that the altered local miRNA changes seen in the RD are reflected in the circulating immune cells, suggesting that miRNAs may contribute to the pathogenesis of psoriasis.     

Circulating miR-142-3p levels in patients with systemic sclerosis

Background. Recently, increased evidence has shown that serum micro (mi)RNA levels are a useful biomarker for the diagnosis, prognosis and therapeutic value of various diseases. However, serum miRNA has not been investigated in patients with systemic sclerosis (SSc), to our knowledge. Aim. To investigate the possibility that serum levels of Homo sapiens miR‐142 stem‐loop (hsa‐miR‐142‐3p), one of the miRNAs regulating the expression of integrin αV, could be a specific disease marker for SSc. Methods. Serum samples were obtained from 61 patients with SSc and 20 healthy controls. Patients with systemic lupus erythematosus (SLE), dermatomyositis (DM) and scleroderma spectrum disorder (SSD), who did not fulfil American College of Rheumatology criteria for SSc but might develop SSc in the future, were included as disease controls in this study. miRNAs were purified from serum, and miR‐142‐3p levels were measured with a quantitative real‐time PCR assay. Results. Serum miR‐142‐3p levels in patients with SSc were significantly higher than in patients with SSD, SLE or DM, and healthy control groups. Patients with increased miR‐142‐3p levels tended to have a short sublingual frenulum. Conclusions. Our data indicate that serum levels of miR‐142‐3p may be elevated specifically in patients with SSc, correlating with the severity of this disease, and may be useful diagnostic markers for the presence of SSc and for the differentiation of SSc from SSD.     

寻常性银屑病患者皮损及外周血miRNA表达谱的研究

目的：探讨寻常性银屑病患者皮损及外周血miRNA表达谱系,寻找表达一致的miRNA。方法用基因芯片技术筛选17例银屑病皮损和外周血miRNA,通过验证筛选出差异性miRNA,探讨其与银屑病皮损和面积严重度指数（PASI）的相关性。结果利用Agilent Human miRNA芯片技术,得出银屑病皮损和外周血中相一致的15个miRNA,经过RT?qPCR技术验证,表达一致的miRNA有7个,其中miR?30e?5p、miR?192?5p、miR?17?3p、miR?1227?5p的表达量与银屑病患者PASI评分呈负相关（P＜0.05）;miR?125b?5p、miR?642a?5p、miR?29a?5p与PASI评分无明显相关性（P＞0.05）。结论寻常性银屑病患者皮损组织和血浆中存在着表达一致的miRNA,这些miRNA有望作为评估银屑病患者病情严重程度的生物标记物之一。     

microRNA-mediated keratinocyte hyperproliferation in psoriasis vulgaris

Background Psoriasis is a chronic inflammatory skin disease characterized by intense proliferation and abnormal differentiation of keratinocytes, although the pathogenesis is still not completely clarified. Objectives We investigated the mechanism of keratinocyte proliferation seen in psoriasis, focusing on microRNA (miRNA). Materials and methods miRNAs were extracted from tissues and sera of psoriasis, atopic dermatitis and healthy control. To determine pathogenic miRNAs, we performed miRNA polymerase chain reaction (PCR) array analysis. The results were confirmed with quantitative real‐time PCR, in situ hybridization, immunohistochemistry, transient transfection of siRNA and inhibitor in cultured keratinocytes and Western blotting. Results PCR array analysis using tissue miRNA demonstrated miR‐424 level was markedly decreased in psoriasis skin in vivo. Protein expression of mitogen‐activated protein kinase kinase 1 (MEK1) or cyclin E1, predicted target genes of miR‐424, was increased in psoriatic skin, although their mRNA levels were not. The transfection of specific inhibitor of miR‐424 in normal human keratinocytes led to upregulation of MEK1 or cyclin E1 protein, and resulted in increased cell proliferation. On the other hand, cell number was significantly decreased when cells were transfected with siRNA for MEK1 or cyclin E1. Furthermore, we first investigated serum miRNA levels in psoriasis. Although not significant, serum miR‐424 concentration tended to be decreased in patients with psoriasis compared with healthy controls. Conclusions Decreased miR‐424 expression and subsequently increased MEK1 or cyclin E1 may play a key role in the pathogenesis of psoriasis. Investigation of the regulatory mechanisms of keratinocyte proliferation by miRNA may lead to new treatments and a disease activity marker.     

MicroRNA expression differs in cutaneous squamous cell carcinomas and healthy skin of immunocompetent individuals

Cutaneous squamous cell carcinoma (cSCC) is one of the most common skin cancers, but the influence of microRNA (miRNA) expression has only been sporadically analysed. We hypothesized that miRNAs are differentially expressed in cSCC and hence influence its development. We therefore isolated total miRNA from well‐differentiated cSCCs and from controls without SCC. Expression analyses of 12 miRNAs showed three significantly differentially expressed miRNAs. We identified a significant upregulation of the miR‐21 and the miR‐31, a proto‐oncogene like miR‐21. While the upregulated expression of miR‐21 has been known for some time, the increased expression of miR‐31 was never shown so clearly. Furthermore, we showed the upregulation of miRNA‐205, which has never been described before. The miR‐205 induces specific keratinocyte migration and could be a characteristic marker for cSCC. It has to be determined in following studies whether these upregulated expressions are specific for cSCC and if so, for which cSCC stages.     

Serum miRNA expression profiles change in autoimmune vitiligo in mice

It is widely believed that non‐segmental vitiligo results from the autoimmune destruction of melanocytes. MicroRNAs (miRNAs), a class of small non‐coding RNAs that negatively regulate gene expression, are involved in the immune cell development and function and regulate the development of autoimmune diseases. Recent studies demonstrate that functional miRNAs can be detected in the serum and serve as biomarkers of various diseases. In the present study, we used a mouse autoimmune vitiligo model, in which melanocyte autoreactive CD4⁺ T cells were adoptively transferred into Rag1^?/? host mice. Serum miRNA expression was profiled in vitiligo developed mice and control mice using TaqMan RT‐PCR arrays. We have found that the expressions of 20 serum miRNAs were changed in vitiligo mice compared to control mice. Three increased miRNAs, miR‐146a, miR‐191, and miR‐342‐3p, were further confirmed by a single TaqMan RT‐PCR. Our findings suggest that miRNAs may be involved in vitiligo development and serum miRNAs could serve as serum biomarkers for vitiligo in mice.     

Risk of subsequent cutaneous malignancy in patients with prior melanoma: a systematic review and meta‐analysis

Melanoma patients are known to be at risk of developing multiple cutaneous (pre‐) malignancies, however, the exact dimensions of these risks are unknown. In this meta‐analysis, risks of developing a melanoma, basal cell carcinoma (BCC) or squamous cell carcinoma (SCC) after a melanoma were investigated. An extensive systematic literature search was conducted (last performed on 18 January 2012). Studies reporting risks, i.e. proportions, standardized incidence ratios (SIR) and cumulative risks (CRs) were included. Fifty, of 233 fully read articles, met selection criteria. Two independent reviewers extracted data on study characteristics and risks measurements. Random‐effects meta‐analyses were used to pool the risk estimates for the three tumour combinations. In melanoma patients, pooled proportions for a subsequent melanoma, BCC or SCC were respectively 3.8% (n = 47), 2.8% (n = 5) and 1.0% (n = 6). The pooled SIRs for a subsequent melanoma, BCC or SCC in melanoma patients were respectively 10.4 (n = 12), 4.6 (n = 2) and 2.8 (n = 2). Mean 20‐year CRs of a subsequent melanoma, BCC or SCC in melanoma patients were respectively 5.4% (n = 3), 14.0% (n = 1) and 4.0% (n = 1). Subgroup analyses showed substantial differences in reported risks between continents and study design. In conclusion, a history of a prior melanoma is a strong predictor for development of a subsequent melanoma (approximately 10‐fold increased risk) and to a lesser extent BCC or SCC. This information could serve as information for health care systems. Further, secondary prevention seems pivotal in this patient group.     

In vivo study for the discrimination of cancerous and normal skin using fibre probe‐based Raman spectroscopy

Raman spectroscopy has proved its capability as an objective, non‐invasive tool for the detection of various melanoma and non‐melanoma skin cancers (NMSC) in a number of studies. Most publications are based on a Raman microspectroscopic ex vivo approach. In this in vivo clinical evaluation, we apply Raman spectroscopy using a fibre‐coupled probe that allows access to a multitude of affected body sites. The probe design is optimized for epithelial sensitivity, whereby a large part of the detected signal originates from within the epidermal layer's depth down to the basal membrane where early stages of skin cancer develop. Data analysis was performed on measurements of 104 subjects scheduled for excision of lesions suspected of being malignant melanoma (MM) (n = 36), basal cell carcinoma (BCC) (n = 39) and squamous cell carcinoma (SCC) (n = 29). NMSC were discriminated from normal skin with a balanced accuracy of 73% (BCC) and 85% (SCC) using partial least squares discriminant analysis (PLS‐DA). Discriminating MM and pigmented nevi (PN) resulted in a balanced accuracy of 91%. These results lie within the range of comparable in vivo studies and the accuracies achieved by trained dermatologists using dermoscopy. Discrimination proved to be unsuccessful between cancerous lesions and suspicious lesions that had been histopathologically verified as benign by dermoscopy.     

Mutations in EXPH5 result in autosomal recessive inherited skin fragility

Several different genes have been implicated in the pathophysiology of inherited blistering skin diseases. Recently, autosomal recessive loss‐of‐function mutations in EXPH5 (encoding exophilin‐5, also known as Slac2‐b, a protein involved in intracellular vesicle transport) were identified in a new mechanobullous disease resembling a form of epidermolysis bullosa simplex (EBS). Here, we searched for mutations in EXPH5 in a 4‐year‐old white boy with EBS in whom initial Sanger sequencing of known genes implicated in intraepidermal skin fragility failed to identify pathogenic mutations. Transmission electron microscopy of rubbed nonlesional patient skin revealed disruption of keratinocytes in the lower epidermis with cytolysis and acantholysis, keratin filament clumping and prominent perinuclear cytoplasmic vesicles, and provided the clue to the candidate gene pathology. Sanger sequencing of genomic DNA showed compound heterozygosity for two new mutations in EXPH5, c.1947dupC (p.Pro649fsPro*11) and c.2249C>A (p.Ser750*). Immunofluorescence microscopy of patient skin showed a complete absence of exophilin‐5 labelling. This case represents the third pedigree with EXPH5 mutations resulting in inherited skin fragility. The clinical and molecular data expand genotype–phenotype correlation in this new form of EBS and demonstrate the important role of exophilin‐5 in keratinocyte cell biology.     

miRNA miR‐17‐92 cluster is differentially regulated in the imiqumod‐treated skin but is not required for imiqumod‐induced psoriasis‐like dermatitis in mice

MicroRNAs (miRNAs) play very important roles in the control of immune cell and keratinocyte development and function and are implicated in skin inflammatory diseases, including psoriasis. miRNA miR‐17‐92 was reported to promote the differentiation of Th1 and Th1 cells and to regulate cell proliferation and apoptosis. Here we showed that imiquimod (IMQ) differentially regulates the expression of miR‐17‐92 cluster in the mouse skin, upregulating miR‐17 and miR‐19 families and downregulating miR‐92. To investigate whether miR‐17‐92 cluster is functionally involved in the psoriasis, we have generated three mutant mice with specific deletion or overexpression of miR‐17‐92 cluster in keratinocytes, or with deletion of miR‐17‐92 cluster in T cells. Interestingly, deletion or overexpression of miR‐17‐92 cluster in keratinocytes, or deletion of miR‐17‐92 in T cells did not significantly affect IMQ‐induced psoriasis‐like dermatitis development in the mutant mice compared with wild‐type littermates. Thus, miRNA miR‐17‐92 cluster may not be a key factor regulating imiqumod‐induced psoriasis‐like dermatitis.     

Evidence on the direct correlation between miR‐31 and IL‐22 axis in IMQ‐induced psoriasis

Psoriatic skin is characterized by a deregulated profile of miRNAs that are contributing in disease development. In this study, we focus on miR‐31, one of the upregulated miRNAs known to promote keratinocytes proliferation and migration. Moreover, miR‐31 expression induction was dependent on a large panel of cytokines including IL‐22. Here, we aimed at investigating the relationship between miR‐31‐5p and IL‐22 axis; and by searching novel molecular target for miR‐31‐5p in keratinocytes. Our data identify a direct correlation between miR‐31‐5p and IL‐22 in psoriasis with Pwp1 as new potential target. These findings confirm the important role of miR‐31 in psoriasis onset and provide a basis for further investigations in miRNAs field in context of skin diseases.     

miR‐137 inhibits proliferation of melanoma cells by targeting PAK2

MicroRNAs (miRNA) are key players in a variety of cancers including malignant melanoma. miR‐137 has been reported to be a tumor suppressor in melanoma and several targets have been identified for this miRNA. We previously developed a novel proteomics technology, ³⁵S in vivo/vitro labelling analysis for dynamic proteomics (SiLAD). Because of its high sensitivity in analysing protein expression rates, SiLAD has the potential to unravel miRNA effects on mRNAs coding for proteins with long half‐lives or high abundance. Using SiLAD, we discovered that miR‐137 significantly downregulated the expression rate of p21‐activated kinase 2 (PAK2) in melanoma cells. Bioinformatics analysis predicted PAK2 as a direct target of miR‐137, which was confirmed by luciferase reporter assay and Western blot analysis. We found that overexpression of miR‐137 inhibited the proliferation of melanoma cells, which could be phenocopied by knockdown of PAK2 using siRNAs. Furthermore, overexpression of PAK2 restored miR‐137‐mediated suppression of cell proliferation. These findings indicate that miR‐137 could inhibit proliferation through targeting PAK2 in melanoma cells.     

Differential expression analysis of miRNA in peripheral blood mononuclear cells of patients with non‐segmental vitiligo

Vitiligo is a common depigmentary skin disease that may follow a pattern of multifactorial inheritance. The essential factors of its immunopathogenesis is thought to be the selective destruction of melanocytes. As a new class of microregulators of gene expression, miRNA have been reported to play vital roles in autoimmune diseases, metabolic diseases and cancer. This study sought to characterize the different miRNA expression pattern in the peripheral blood mononuclear cells (PBMC) of patients with non‐segmental vitiligo (NSV) and healthy individuals and to examine their direct responses to thymosin α1 (Tα1) treatment. The miRNA expression profile in the PBMC of patients with NSV was analyzed using Exiqon's miRCURY LNA microRNA Array. The differentially expressed miRNA were validated by real‐time quantitative polymerase chain reaction. We found that the expression levels of miR‐224‐3p and miR‐4712‐3p were upregulated, and miR‐3940‐5p was downregulated in the PBMC. The common clinical immune modulator Tα1 changed the miRNA expression profile. Our analysis showed that differentially expressed miRNA were associated with the mechanism of immune imbalance of vitiligo and that Tα1 could play an important role in changing the expression of these miRNA in the PBMC of patients with NSV. This study provided further evidence that miRNA may serve as novel drug targets for vitiligo therapeutic evaluation.     

Lipid profile in actinic keratosis and basal cell carcinoma

BACKGROUND: The lipid content of the skin and its changes are important in the pathogenesis of many disorders affecting the skin, particularly actinic keratosis (AK) and basal cell carcinoma (BCC). METHODS: Cholesterol, phospholipid, triglyceride, and total lipid levels were studied in paired lesional (AK and BCC) and nonlesional intact skin of 13 patients with AK and 12 patients with BCC. Serum concentrations of the same lipid fractions studied in the skin were investigated in AK and BCC patients and in 11 healthy, age-matched controls. RESULTS: Levels of all lipid fractions were increased in both AK and BCC skin. When AK and BCC skin were compared with each other, a significant increase in phospholipids (p < 0.02) and total lipids (p < 0.01) was found in BCC. Serum cholesterol (p < 0.001), phospholipid (p < 0.001), triglyceride (p < 0.05), and total lipid (p < 0.001) concentrations of AK patients were significantly higher than those of the control group. When BCC and controls were compared, a significant increase in phospholipids and total lipids (p < 0.001) was seen. Serum cholesterol in BCC patients was significantly lower (p < 0.001) and serum phospholipid levels were significantly higher (p < 0.05) than those in the AK group. CONCLUSIONS: An increase in the metabolically active serum phospholipid fraction is reflected in elevated neoplastic tissue phospholipid. This produces altered proportions between lipid fractions in tumorous areas and may result in changes in the intact nature of the cellular membrane, spread, and malignant proliferation.