Recent progress in studies of miRNA and skin diseases

miRNA is a family of small non‐coding RNA that consists of 22 nucleotides on average. miRNA are implicated in various cellular activities such as cell proliferation or migration via the modulation of gene expression, and also are linked to the pathogenesis of human diseases. This paper reviews recent research progress about the contribution of miRNA to the pathogenesis of various skin diseases, and possible application of miRNA as the disease markers in each disease. For example, downregulated miR‐424‐5p in psoriatic skin causes the overexpression of MEK1 and cyclin E1 in psoriatic keratinocytes, resulting in the keratinocyte overgrowth and hyperproliferation seen in the disease. Although there was no significant difference in the serum miR‐424‐5p levels between psoriasis patients and healthy controls, serum miR‐1266‐5p levels were significantly upregulated in psoriasis patients, and showed weak and inverse correlation with disease activity. Furthermore, combination of serum levels of miR‐146a‐5p and ‐203a‐3p was more reliable to distinguish psoriasis patients and normal subjects, than each miRNA alone. Hair shaft miR‐424‐5p levels were significantly higher in psoriasis patients than normal subjects, while hair root miR‐19a‐3p levels in psoriasis patients were inversely correlated with the duration between symptom onset and the first visit to the hospital. Future researches of miRNA will enable the advances of their clinical applications including the clarification of pathogenesis, disease markers and novel treatments.     

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-125b, a microRNA downregulated in psoriasis, modulates keratinocyte proliferation by targeting FGFR2

MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play important roles in the regulation of gene expression. We previously identified a characteristic miRNA expression profile in psoriasis, distinct from that of healthy skin. One of the most downregulated miRNAs in psoriasis skin was microRNA-125b (miR-125b). In this study, we aimed to identify the potential role(s) of miR-125b in psoriasis pathogenesis. In situ hybridization results showed that the major cell type responsible for decreased miR-125b levels in psoriasis lesions was the keratinocyte. Overexpression of miR-125b in primary human keratinocytes suppressed proliferation and induced the expression of several known differentiation markers. Conversely, inhibition of endogenous miR-125b promoted cell proliferation and delayed differentiation. Fibroblast growth factor receptor 2 (FGFR2) was identified as one of the direct targets for suppression by miR-125b by luciferase reporter assay. The expression of miR-125b and FGFR2 was inversely correlated in both transfected keratinocytes and in psoriatic skin. Knocking down FGFR2 expression by siRNA suppressed keratinocyte proliferation, but did not enhance differentiation. Altogether, our results demonstrate a role for miR-125b in the regulation of keratinocyte proliferation and differentiation, partially through the regulation of FGFR2. Loss of miR-125b in psoriasis skin may contribute to hyperproliferation and aberrant differentiation of keratinocytes.     

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.     

Small-interfering RNA targeted at antiapoptotic mRNA increases keratinocyte sensitivity to apoptosis

Background Gene silencing RNA interference technology concentrates on downregulation of gene expression using specific double‐stranded RNA molecules (small‐interfering RNA, siRNA), which induce the degradation of complementary mRNA. SiRNA therapeutics is currently being tested in several clinical trials. Skin disorders are an attractive target for siRNA‐based technology because effective agents can be delivered by topical application. In several inflammatory skin disorders, the barrier function of the skin is markedly impaired and this may increase the delivery efficiency. Psoriasis is a very common skin disorder. The characteristics of this disorder are abnormal keratinocyte proliferation and inflammation. Keratinocytes from psoriatic epidermis express much higher levels of the antiapoptotic protein, Bcl‐xL, compared with normal keratinocytes. Insulin‐like growth factor 1 receptor (IGF‐1R) plays a major role in cell growth, differentiation and apoptosis in many cell types, including keratinocytes and IGF‐1R activation plays an important role in the pathogenesis of psoriasis. Keratinocytes from patients with psoriasis are more susceptible to IGF‐1‐stimulated proliferation compared with normal keratinocytes. IGF‐1R is expressed by proliferating basal and suprabasal keratinocytes and is more abundant in psoriatic lesions. Objectives To prove the validity of IGF‐1R and Bcl‐xL as useful targets for siRNA‐based therapeutics and to deliver siRNA selectively and efficiently to primary human keratinocytes; this is a primary essential step in the development of siRNA. Methods Primary normal human keratinocytes were transfected with various siRNA molecules, and transfection efficiency was monitored by fluorescent labelling. Cell growth and apoptosis induction were evaluated in the transfected cells. Results We were able to deliver efficiently siRNA targeting Bcl‐xL or IGF‐1R to primary human keratinocyte cultures. We also showed that siRNAs targeting Bcl‐xL and IGF‐1R induce growth inhibition, apoptosis and increased sensitivity to ultraviolet B in keratinocytes. Conclusions The present findings demonstrate that Bcl‐xL and IGF‐1R are valid, important targets for siRNA‐based technology directed at the suppression of keratinocyte hyperproliferation.     

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.     

Genetic polymorphisms altering microRNA activity in psoriasis – a key to solve the puzzle of missing heritability?

Psoriasis is a chronic immune‐mediated skin disease in which the balance in the interplay of immune cells and keratinocytes is disturbed. MicroRNAs (miRNAs) are endogenous small regulatory RNAs that stabilize cellular phenotypes and fine‐tune signal transduction feedback loops through the regulation of gene networks. Through the regulation of their multiple target genes, miRNAs regulate the development of inflammatory cell subsets and have a significant impact on the magnitude of inflammatory responses. Since the discovery of deregulated miRNA expression in psoriasis, we have learned that they can regulate differentiation, proliferation and cytokine response of keratinocytes, activation and survival of T cells and the crosstalk between immunocytes and keratinocytes through the regulation of chemokine production. In recent years, it became apparent that genetic polymorphisms in miRNA genes and/or in miRNA binding sites of target genes can affect miRNA activity and contribute to disease susceptibility. Psoriasis has a strong genetic background; however, the contribution of genetic variants involving miRNAs is largely unexplored in psoriasis. We propose that changes in miRNA‐mediated gene regulation may be a major contributor to the disturbed balance in immune regulation that results in chronic skin inflammation. In this viewpoint essay, we focus on the emerging new aspects of the role of miRNAs in psoriasis and propose that genetic polymorphisms that affect miRNA activity might be important in the pathogenesis of psoriasis.     

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。     

The anti‐apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non‐coding RNA,PRINS

Please cite this paper as: The anti‐apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non‐coding RNA, PRINS. Experimental Dermatology 2010; 19: 269–278. Abstract: Psoriasis Susceptibility‐Related RNA Gene Induced by Stress (PRINS) is a non‐coding RNA overexpressed in lesional and non‐lesional psoriatic epidermis and induced by stress. Its function in healthy and psoriatic skin is still not known. Here, we report that PRINS regulates G1P3, a gene with anti‐apoptotic effects in keratinocytes. siRNA‐mediated inhibition of PRINS gene resulted in altered cell morphology and gene expression alterations, as demonstrated in a microarray experiment. One of the genes regulated by PRINS ncRNA was G1P3, an interferon‐inducible gene with anti‐apoptotic effects in cancer cells. Interestingly, we found that G1P3 was 400‐fold upregulated in hyperproliferative lesional and ninefold upregulated in non‐lesional psoriatic epidermis compared to healthy epidermis. In vitro, G1P3 protein levels were highest in proliferating keratinocytes and siRNA‐mediated downregulation of G1P3 resulted in increased cell apoptosis. These data indicate that G1P3 inhibits spontaneous keratinocyte apoptosis and hence its high expression in psoriatic skin may contribute to the development of psoriatic lesions. We hypothesize that the deregulation of the PRINS ncRNA may contribute to psoriasis and results in decreased sensitivity to spontaneous keratinocyte apoptosis via the regulation of G1P3.     

Overexpression of connexin26 in the basal keratinocytes reduces sensitivity to tumor promoter TPA

Please cite this paper as: Overexpression of connexin26 in the basal keratinocytes reduces sensitivity to tumor promoter TPA. Experimental Dermatology 2010; 19: 633–640. Abstract: Connexin 26 is important in keratinocyte proliferation, differentiation and skin pathologies. Cx26 is barely expressed in normal adult epidermis, but its expression is induced during wound healing, psoriasis, and skin hyperplasia stimulated by tumor promoters. In hyperplastic proliferating epidermis, Cx26 is co‐expressed with Cx43 typical for basal and suprabasal keratinocytes. As Cx26 and Cx43 can not form permeable gap junctions, their co‐expression may alter the gap junctional communication between keratinocytes and induce proliferation. To test the effect of persistent co‐expression of Cx26 and Cx43 in epidermis, we generated transgenic mice using keratin5 promoter to target Cx26 to basal Cx43‐positive keratinocytes. We evaluated the effect of ectopic Cx26 on keratinocyte proliferation and differentiation in normal and 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA)‐treated skin. The ectopic Cx26 expression in epidermis did not significantly affect skin development, keratinocyte differentiation and proliferation in newborn and adult skin. Unexpectedly, the proliferative effect of tumor promoter TPA was strongly decreased in epidermis of K5.Cx26 transgenics. This correlated with significant down‐regulation of TPA‐induced activity of protein kinase C (PKC) in K5.Cx26 mice.     

MiR‐744‐3p regulates keratinocyte proliferation and differentiation via targeting KLLN in psoriasis

Psoriasis is a chronic inflammatory disease, and microRNAs have been reported to regulate the pathogenesis of psoriasis. Up‐regulated miR‐744‐3p level was identified to associate with psoriasis while the precise functions of miR‐744‐3p in psoriasis were not well‐elucidated. We first confirmed the up‐regulation of miR‐744‐3p in psoriasis by measuring its expression level in psoriatic samples. We explored the roles of miR‐744‐3p on keratinocytes proliferation and differentiation. We searched the targets of miR‐744‐3p and evaluated the roles of one target, KLLN on keratinocytes proliferation and differentiation. We confirmed the up‐regulation of miR‐744‐3p in psoriatic samples. MiR‐744‐3p promoted keratinocytes proliferation while inhibited differentiation. MiR‐744‐3p targeted KLLN and overexpression of miR‐744‐3p resulted in decreased expression of KLLN. Overexpression of KLLN prevented the effects of miR‐744‐3p on keratinocytes proliferation and differentiation. MiR‐744‐3p regulated the proliferation and differentiation of keratinocytes through targeting KLLN in psoriasis.     

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.     

Mapping of a novel locus for an autosomal recessive form of palmoplantar keratoderma on chromosome 3q27.2‐q29

Many exogenous factors including excessive alcohol consumption have been associated with psoriasis, but the underlying mechanisms still remain elusive. Drinking worsens therapeutic compliance, and decreases the efficacy and increases the toxicity of systemic antipsoriatic treatments. Excess alcohol intake results in compromised immunity and increased risk of infections, but alcohol can induce proinflammatory cytokine production in various cell types and can increase mitogen‐derived lymphocyte proliferation and lymphocyte activation. As we have previously reported, alcohol and one of its metabolites, acetone, induce keratinocyte proliferation and increase the mRNA levels of genes characteristic for proliferating keratinocytes, such as α5 integrin, cyclin D1 and keratinocyte growth factor receptor. Recently the correlation between blood and skin ethanol levels in humans was determined by a transdermal alcohol monitoring device, against the ‘gold standard’ breath alcohol readings. Based on transdermal alcohol measurements it can be concluded that cutaneous alcohol concentrations can reach levels that induce proinflammatory cytokine production and lymphocyte and keratinocyte proliferation in vitro. It is expected that the development of methodologies measuring transdermal ethanol will provide additional tools to evaluate how alcohol influences skin physiology and different dermatological conditions including psoriasis. Our review focuses on the possible link between alcohol misuse and psoriasis, particularly on the possible role of cutaneous ethanol in precipitating the disease.     

APOBEC3 regulates keratinocyte differentiation and expression of Notch3

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐like (APOBEC) family consists of deaminases. Some isozymes of APOBEC3 are induced upon human papillomavirus infection or development of psoriasis skin lesions. However, the involvement of APOBEC3 in keratinocyte differentiation has not been addressed. We herein sought to evaluate the roles of APOBECs in mouse primary keratinocyte differentiation. We found that expression levels of APOBEC1 and APOBEC3 were increased during calcium‐induced keratinocyte differentiation. Unexpectedly, however, the expression levels of keratinocyte differentiation markers keratin 1/10, involucrin, loricrin and filaggrin were higher in keratinocytes treated with APOBEC3 siRNAs than in those treated with control RNAs. In addition, the treatment of keratinocytes with APOBEC3 siRNAs increased the gene expression levels of Notch3, a master regulator of keratinocyte differentiation. Moreover, calcium‐induced increase in Notch3 expression and keratinocyte differentiation were impaired by transfection with an APOBEC3 expression plasmid. Furthermore, co‐treatment with Notch3 siRNAs reduced the APOBEC3 siRNA‐mediated upregulation of Notch3 expression and in part attenuated the increased expression levels of keratinocyte differentiation markers. These results suggest that APOBEC3 is induced upon keratinocyte differentiation and negatively regulates the keratinocyte differentiation in part by its inhibitory role for Notch3 expression.     

Induction of senescence pathways in Kindler syndrome primary keratinocytes

Background Individuals with Kindler syndrome (KS) have loss‐of‐function mutations in the FERMT1 gene that encodes the focal adhesion component kindlin‐1. The major clinical manifestation of KS is epidermal atrophy (premature skin ageing). This phenotypic feature is thought to be related to the decreased proliferation rate of KS keratinocytes; nevertheless, molecular mediators of such abnormal behaviour have not been fully elucidated. Objectives To investigate how kindlin‐1 deficiency affects the proliferative potential of primary human keratinocytes. Methods  We serially cultivated nine primary KS keratinocyte strains until senescence and determined their lifespan and colony‐forming efficiency (CFE) at each serial passage. The expression of molecular markers of stemness and cellular senescence were investigated by immunoblotting using cell extracts of primary keratinocyte cultures from patients with KS and healthy donors. In another set of experiments, kindlin‐1 downregulation in normal keratinocytes was obtained by small interfering RNA (siRNA) technology. Results We found that KS keratinocytes exhibited a precocious senescence and strongly reduced clonogenic potential. Moreover, KS cultures showed a strikingly increased percentage of aborted colonies (paraclones) already at early passages indicating an early depletion of stem cells. Immunoblotting analysis of KS keratinocyte extracts showed reduced levels of the stemness markers p63 and Bmi‐1, upregulation of p16 and scant amounts of hypophosphorylated Rb protein, which indicated cell cycle‐arrested status. Treatment of normal human primary keratinocytes with siRNA targeting kindlin‐1 proved that its deficiency was directly responsible for p63, Bmi‐1 and pRb downregulation and p16 induction. Conclusions Our data directly implicate kindlin‐1 in preventing premature senescence of keratinocytes.     

sprouty4蛋白对角质形成细胞增殖及分化的影响

 目的:探证sprouty4(SPRY4)蛋白对角质形成细胞增殖及分化的影响。方法：以人永生化表皮细胞株HaCaT细胞作为实验对象,实验组HaCaT细胞采用基因敲降技术进行SPRY4蛋白抑制物的转染,对照组HaCaT细胞不作任何处理。运用实时荧光定量PCR技术（RT-qPCR）检测实验组与对照组中sprouty4蛋白对HaCaT细胞分化指标Involucrin、CK1与CK10的影响,运用CCK-8实验检测HaCaT细胞的增殖功能。结果：RT-qPCR结果显示,实验组HaCaT细胞中SPRY4基因敲降成功,敲降率约为94.75%。与对照组相比,实验组HaCaT细胞中分化指标Involucrin、CK1与CK10表达水平降低;CCK-8实验结果显示,与对照组相比,实验组HaCaT细胞增殖能力增强。结论：SPRY4蛋白表达下降对细胞增殖起到促进作用,对细胞分化起到抑制作用。     

Effects of a 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitor and low‐density lipoprotein on proliferation and migration of keratinocytes

Background Keratinocytes can obtain cholesterol either by de novo synthesis or by extraction, primarily from low‐density lipoprotein (LDL). LDL is internalized following binding to the LDL receptor (LDLR). Because LDLR is expressed at a higher level in the cells of the basal layer of the epidermis, it might be assumed that LDLR upregulation is associated with keratinocyte proliferation. However, the effect of LDLR stimulation on keratinocyte function remains unclear. Objectives To investigate the effects and mechanism of action of pitavastatin and effects of LDL on proliferation and migration of keratinocytes. Methods Pitavastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, was used to induce upregulation of LDLR. LDLR expression was evaluated by immunofluorescence staining, fluorescence‐activated cell sorting, immunohistochemical staining and real‐time polymerase chain reaction (PCR). HaCaT cells and normal human keratinocytes (NHKs) were used for evaluation of migration. 5‐Bromo‐2′‐deoxyuridine incorporation was used to evaluate keratinocyte proliferation and differentiation. C57BL6 mice were used for in vivo evaluation of the effect of topical pitavastatin or lovastatin. Results Pitavastatin was most effective in LDLR induction at a concentration of 1 μmol L^?1 in NHKs. Real‐time PCR showed that pitavastatin significantly increased LDLR and liver X receptor (LXR) β mRNA expression in these cells. Similar results were obtained in vivo. However, pitavastatin had no effect on the migration of NHKs. After the addition of LDL and/or mevalonate concomitantly with pitavastatin to NHK cultures, or topical application of pitavastatin on mouse skin, keratinocyte proliferation was significantly increased. Conclusions Pitavastatin significantly upregulates LDLR in both NHKs and C57BL6 mouse skin, resulting in increased keratinocyte proliferation. LXRβ may be involved in the pitavastatin‐induced keratinocyte proliferation.