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.     

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.     

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。     

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.     

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。     

microRNA在炎症性皮肤病中作用的研究进展

【摘要】 microRNA（miRNA）是一类转录后调控基因表达的非编码RNA分子,参与皮肤各种病理生理过程。近年来,miRNA表达谱的变化已被报道与部分炎症性皮肤病相关,例如miR-203、miR-146a、miR-21在银屑病皮损中表达上调;miR-155、miR-146a在特应性皮炎皮损中表达上调;miR-21、miR-223、miR-142-3p、miR142-5p在过敏性接触性皮炎皮损表达上调;而miR-146a、miR-155在系统性红斑狼疮患者外周血表达下调;miR-223在皮肌炎皮损中表达下调等。本文综述miRNA与部分炎症性皮肤病发生、发展之间的联系。     

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在人皮肤黑素瘤与色素痣中共同差异表达的研究

目的 筛选人皮肤黑素瘤组织中表达的已知hsa-microRNA。方法 采用SBC microRNA芯片技术,将6例人皮肤黑素瘤组织总RNA分别与9例健康人色素痣总RNA混合物对比,筛选已知的468条hsa-microRNA表达情况。用qPCR分别检测这6例人皮肤黑素瘤组织中各候选hsa-microRNA的表达。将芯片和qPCR两种方法检测结果一致的候选hsa-microRNA确定为有意义的共同差异表达hsa-microRNA。结果 2倍以上差异表达miRNA占所检测miRNA的12.18% ～ 86.33%,5倍以上差异表达miRNA占1.28% ～ 19.02%,10倍以上差异表达miRNA占0.43% ～ 5.34%。 此6例人皮肤黑素瘤中miRNA-21 明显上调表达,miRNA-320和miRNA-494明显下调表达。结论 人皮肤黑素瘤组织中miRNA-21明显上调表达,miRNA-320和miRNA-494明显下调表达。     

Expression analysis of multiple microRNAs in each patient with scleroderma

In this study, we compared expression pattern of multiple microRNAs in individual patient with scleroderma with that in normal subject. Serum levels of six microRNAs (miR‐7 g, miR‐21, miR‐29b, miR‐125, miR‐145 and miR‐206) were evaluated using real‐time PCR in 15 patients with scleroderma and 15 normal subjects. While levels of the six microRNAs were similar between the two groups, we found significant difference in the ranks between miRNAs in patients with scleroderma. Additionally, levels of let‐7 g and miR‐125b showed strong and significant correlation in normal subjects, but not in patients with scleroderma. Thus, miRNA expression pattern may be different in patients with scleroderma. We also found the combination of serum levels of miR‐206 and miR‐21 was more useful in distinguishing patients with scleroderma from normal subjects than either miR‐206 or miR‐21 alone. Our study is the first to demonstrate different expression profiles of multiple microRNAs in each patient with scleroderma and examine its clinical significance.     

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.     

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.     

Unique microRNAs appear at different times during the course of a delayed‐type hypersensitivity reaction in human skin

Diphencyprone (DPCP) is a hapten that induces delayed‐type hypersensitivity (DTH) reactions. MicroRNAs (miRNAs) are short non‐coding RNAs that negatively regulate gene expression and have been implicated in various inflammatory skin diseases, but their role in DTH reactions is not well understood. We generated global miRNA expression profiles (using next‐generation sequencing) of DPCP reactions in skin of seven healthy volunteers at 3, 14 and 120 days after challenge. Compared to placebo‐treated sites, DPCP‐challenged skin at 3 days (peak inflammation) had 127 miRNAs significantly deregulated. At 14 days (during resolution of inflammation), 43 miRNAs were deregulated and, at 120 days (when inflammation had completely resolved), six miRNAs were upregulated. While some miRNAs have been observed in psoriasis or atopic dermatitis, most of the deregulated miRNAs have not yet been studied in the context of skin biology or immunology. Across the three time points studied, many but not all miRNAs were uniquely expressed. As various miRNAs may influence T cell activation, this may indicate that the miRNAs exclusively expressed at different time points function to promote or resolve skin inflammation, and therefore, may inform on the paradoxical ability of DPCP to treat both autoimmune conditions (alopecia areata) and conditions of ineffective immunity (melanoma).     

The contribution of feedback loops between miRNAs,cytokines and growth factors to the pathogenesis of psoriasis

The present review describes in detail the existent data regarding feedback loops between miRNAs and cytokines or growth factors in the psoriatic inflammation. We have chosen to describe the roles of miR‐31, miR‐21, miR‐146a, miR‐155, miR‐197 and miR‐99a in this process. This choice derives from the fact that among around 250 miRNAs being altered in the psoriatic lesion, the comprehensive functional role was described only in those detailed above. In addition, considering the molecular targets and the pathways, which may possibly be regulated by those miRNAs, it seems that they may be chosen as preferred targets for the therapy of psoriasis.     

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.