Wakana Nakayama

microRNA-7 down-regulation mediates excessive collagen expression in localized scleroderma.

Localized scleroderma (LSc), a connective tissue disorder restricted to the skin and subcutaneous tissue, is characterized by skin fibrosis due to an excessive deposition of types I collagen. The mechanism of such fibrosis is still unknown, but epigenetics may play some roles in the excessive collagen expression. In the present study, we investigated the mechanism of fibrosis seen in LSc, focusing on microRNA (miRNA). miRNA expression was determined by PCR array, real-time PCR, and in situ hybridization. The function of miRNA was evaluated using specific inhibitor. Immunoblotting was performed to detect α2(I) collagen protein. PCR array analysis using tissue miRNA demonstrated miR-7 level was significantly decreased in LSc skin as well as keloid tissue compared to normal skin in vivo. In situ hybridization also showed miR-7 expression in dermal fibroblasts was decreased in LSc dermis. The transfection of specific inhibitor for miR-7 into cultured normal dermal fibroblasts resulted in the up-regulation of α2(I) collagen protein in vitro. Also, the serum levels of miR-7 were significantly decreased in LSc patients compared with healthy controls, but serum miR-29a levels not. Systemic or local down-regulation of miR-7 may contribute to the pathogenesis of LSc via the overexpression of α2(I) collagen, and serum miR-7 may be useful as a disease marker. Investigation of the regulatory mechanisms of LSc by miRNA may lead to new treatments by the transfection into the lesional skin of this disease.

Detection of hair-microRNAs as the novel potent biomarker: Evaluation of the usefulness for the diagnosis of scleroderma

BACKGROUND Serum microRNA levels are known as useful biomarkers for various diseases. Recent publication has indicated the existence of microRNAs in hair roots and hair shafts. OBJECTIVE In this study, we evaluated several methods for the extraction of hair microRNAs, and their usefulness for the diagnosis of scleroderma. METHODS A single hair root and 5 pieces of hair shafts were obtained from the occiput of each individual of 11 scleroderma patients and 13 normal subjects at the time of serum sampling. microRNA extraction from sera or hair roots was performed with commercially available kits. microRNAs were extracted from hair shafts using four different methods. microRNA expression was evaluated by PCR array and real-time PCR. RESULTS We demonstrated microRNAs in hair roots and hair shafts were detectable and quantitative using our method. We found the difference of microRNA levels in hair roots and hair shafts obtained from different places of head in each individual were within 2-fold, indicating the reproducibility of hair microRNA levels by our method. PCR array revealed microRNAs from sera, hair roots and hair shafts have different expression pattern, and can be independent biomarkers. Serum and hair root miR-196a levels were not significantly changed in scleroderma patients, while we found miR-196a levels in hair shafts were significantly decreased in scleroderma patients compared to those in normal subjects (p<0.05). CONCLUSION Hairs are more accessible than sera among human samples. microRNAs levels in hair roots or hair shafts may become effective and independent biomarkers.

Long non-coding RNA TSIX is upregulated in scleroderma dermal fibroblasts and controls collagen mRNA stabilization

Long non‐coding RNAs (lncRNAs) are thought to have various functions other than RNA silencing. We tried to evaluate the expression of lncRNAs in patients with systemic sclerosis (SSc) and determined whether lncRNAs controls collagen expression in dermal fibroblasts. lncRNA expression was determined by real‐time PCR and in situ hybridization. Protein and mRNA levels of collagen were analysed using immunoblotting and real‐time PCR. We found TSIX, one of the lncRNAs, was overexpressed in SSc dermal fibroblasts both in vivo and in vitro, which was inhibited by the transfection of transforming growth factor (TGF)‐β1 siRNA. TSIX siRNA reduced the mRNA expression of type I collagen in normal and SSc dermal fibroblasts, but not the levels of major disease‐related cytokines. In addition, TSIX siRNA significantly reduced type I collagen mRNA stability, but not protein half‐lives. Furthermore, we first investigated serum lncRNA levels in patients with SSc, and serum TSIX levels were significantly increased in SSc patients. TSIX is a new regulator of collagen expression which stabilizes the collagen mRNA. The upregulation of TSIX seen in SSc fibroblasts may result from activated endogenous TGF‐β signalling and may play a role in the constitutive upregulation of collagen in these cells. Further studies on the regulatory mechanism of tissue fibrosis by lncRNAs in SSc skin lead to a better understanding of the pathogenesis, new diagnostic methods by their serum levels and new therapeutic approaches using siRNAs.

Serum levels of soluble CD163 in patients with systemic sclerosis

Macrophages may play a role in the pathogenesis of systemic sclerosis (SSc), and CD163-positive M2 macrophages are potentially important source for fibrosis-inducing cytokines. However, no link between M2 macrophages and SSc has been established. The aim is to evaluate the possibility that serum levels of soluble CD163 (sCD163) can be a useful marker for SSc, reflecting M2 activation of macrophages in this disease. Serum sCD163 levels of 43 patients with SSc, 10 patients with scleroderma spectrum disorder (SSD), and 12 healthy controls were measured with specific enzyme-linked immunosorbent assays. SSc patients had significantly higher serum sCD163 levels than healthy controls. The sCD163 levels in SSD patients were higher than healthy controls and lower than SSc patients. Significantly higher right ventricular systolic pressure and lower % DLco levels, and shorter duration of disease were seen in SSc patients with elevated serum sCD163 levels than those with normal levels. These results suggest that sCD163 levels may be increased in proportion to the progression of this disease, indicating the involvement of CD163 in the pathogenesis of SSc. Furthermore, serum sCD163 levels may be a marker of pulmonary hypertension at the early stage in patients with SSc.

Hair miR-29a levels are decreased in patients with scleroderma

In the present study, we evaluated the possibility that we can utilize hair shaft miR‐29a levels as disease marker of scleroderma. Hair samples were obtained from 20 scleroderma patients, five dermatomyositis patients and 13 controls. microRNAs were purified from hairs as well as skins or sera, and miR‐29a levels were measured with quantitative real‐time polymerase chain reaction. Mean hair miR‐29a levels in scleroderma patients were significantly lower than those in control subjects or dermatomyositis, while expression levels of hair shaft marker keratin 34 were similar among them. There was no strong correlation among the miR‐29a levels in the hair, skin and serum of each patient, suggesting that hair microRNAs can be independent biomarkers. We found scleroderma patients with decreased miR‐29a levels had contracture of the phalanges at a significantly higher prevalence than those without. To confirm the clinical usefulness of hair microRNAs, large‐scale researches are needed in the future.

Decreased miR-7 Expression in the Skin and Sera of Patients with Dermatomyositis

Expression of microRNA (miRNA) in the skin in dermatomyositis has not previously been studied in detail. In this study, we performed miRNA array analysis using miRNAs purified from dermatomyositis-involved skin and normal skin, and found that several miRNAs were up- or down-regulated in dermatomyositis skin. Among them, we focused on miR-7, one of the most down-regulated miRNAs in dermatomyositis skin. Total miRNAs were purified from serum, and hsa-miR-7 levels were measured with quantitative real-time PCR using the specific primer. Serum levels of miR-7 were significantly decreased in patients with dermatomyositis compared with normal subjects or patients with other autoimmune diseases. Thus, serum miR-7 levels might be a possible diagnostic marker for dermatomyositis. Clarifying the up- or down-stream events of down-regulated miR-7 in patients with dermatomyositis may lead to further understanding of the disease and a new therapeutic approach.

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.

Down-regulation of miR-223 contributes to the formation of Gottron's papules in dermatomyositis via the induction of PKCε

BACKGROUND Dermatomyositis (DM) is characterized by skin manifestations accompanying and preceding muscle weakness. Gottrons papules, one of the skin manifestations, are of great diagnostic value because they are specific to DM. However, the pathogenesis of Gottrons papules remains unclear. OBJECTIVES We investigated the expression pattern of miRNAs in Gottrons papules of DM patients and evaluated the possibility that miRNAs play a role in its pathogenesis. MATERIALS AND METHODS miRNAs were extracted from skin tissues and sera of patients with DM, clinically amyopathic DM (CADM) and healthy controls. To identify pathogenic miRNAs, we performed miRNA PCR array analysis. The results were confirmed by in situ hybridization, immunohistochemistry, immunoblotting and transient transfection of siRNAs or miRNA inhibitors. RESULTS PCR array analysis using tissue miRNAs demonstrated the miR-223 level was markedly decreased in Gottrons papules of DM and CADM in vivo, but not in psoriasis skin. The protein expression of PKCɛ, a predicted target of miR-223, was increased in DM/CADM skin. The transfection of a specific inhibitor of miR-223 in keratinocytes led to up-regulation of the PKCɛ protein, and resulted in increased cell proliferation. On the other hand, cell numbers were significantly decreased when cells were transfected with siRNA for PKCɛ. The serum miR-223 concentration was decreased in DM/PM patients, particularly in CADM patients, compared with healthy controls. CONCLUSIONS A decreased miR-223 expression and the subsequently increased PKCɛ levels may therefore play a key role in the pathogenesis of Gottrons papules.

The miR-146a rs2910164 C/G polymorphism is associated with telangiectasia in systemic sclerosis.

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CD163 expression is increased in the involved skin and sera of patients with systemic lupus erythematosus

OBJECTIVE To investigate the relationship between M2 macrophages and disease activity in SLE. METHODS The expression of CD163, an M2 macrophage marker, in skin specimens was evaluated by immunohistochemistry. Quantitative real-time polymerase chain reaction was performed to determine mRNA expression of CD163 in the skin section. Serum levels of soluble CD163 were measured in 20 SLE patients and 12 healthy controls with specific enzyme-linked immunosorbent assays. RESULTS The number of CD163-positive M2 macrophages and the mRNA levels of CD163 in the skin of SLE patients was significantly increased. SLE patients had significantly higher serum sCD163 levels than healthy controls. By analysis of the association between serum sCD163 levels and the clinical/laboratory features, we found that patients with elevated serum sCD163 levels showed significantly higher levels of anti-double-strand-DNA antibodies and higher prevalence of leukopenia than those with normal levels. CONCLUSION Infiltration of CD163-positive M2 macrophages was increased in SLE skin and sCD163 levels were increased in SLE sera. A systemic increase of sCD163 as well as local overexpression of CD163 in SLE patients indicate that M2 macrophages may play a role in the pathogenesis of SLE. Serum sCD163 levels may be a useful marker for disease severity in patients with SLE.