Zhongzhi Wang

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.

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.

NUP160-SLC43A3 Is a Novel Recurrent Fusion Oncogene in Angiosarcoma

Angiosarcoma is a malignant vascular tumor originating from endothelial cells of blood vessels or lymphatic vessels. The specific driver mutations in angiosarcoma remain unknown. In this study, we investigated this issue by transcriptome sequencing of patient-derived angiosarcoma cells (ISO-HAS), identifying a novel fusion gene NUP160-SLC43A3 found to be expressed in 9 of 25 human angiosarcoma specimens that were examined. In tumors harboring the fusion gene, the duration between the onset of symptoms and the first hospital visit was significantly shorter, suggesting more rapid tumor progression. Stable expression of the fusion gene in nontransformed human dermal microvascular endothelial cells elicited a gene-expression pattern mimicking ISO-HAS cells and increased cell proliferation, an effect traced in part to NUP160 truncation. Conversely, RNAi-mediated attenuation of NUP160 in ISO-HAS cells decreased cell number. Confirming the oncogenic effects of the fusion protein, subcutaneous implantation of NUP160-SLC43A3-expressing fibroblasts induced tumors resembling human angiosarcoma. Collectively, our findings advance knowledge concerning the genetic causes of angiosarcoma, with potential implications for new diagnostic and therapeutic approaches.

EBI3 Downregulation Contributes to Type I Collagen Overexpression in Scleroderma Skin

IL-12 family cytokines are implicated in the pathogenesis of various autoimmune diseases, but their role in the regulation of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) remain to be elucidated. Among the IL-12 family members, IL-35 decreases type I collagen expression in cultured dermal fibroblasts. IL-35 consists of p35 and EBI3 subunits, and EBI3 alone could downregulate the protein and mRNA expression of type I or type III collagen in the presence or absence of TGF-β costimulation. We found that collagen mRNA stability was reduced by EBI3 via the induction of miR-4500. The IL-35 levels in the sera or on the surface of T cells were not altered in SSc patients, while EBI3 expression was decreased in the keratinocytes of the epidermis and regulatory T cells of the dermis in SSc skin compared with normal skin, which may induce collagen synthesis in SSc dermal fibroblasts. We also found that gp130, the EBI3 receptor, was expressed in both normal and SSc fibroblasts. Moreover, we revealed that EBI3 supplementation by injection into the skin improves mice skin fibrosis. Decreased EBI3 in SSc skin may contribute to an increase in collagen accumulation and skin fibrosis. Clarifying the mechanism regulating the extracellular matrix expression by EBI3 in SSc skin may lead to better understanding of this disease and new therapeutic strategies using ointment or microinjection of the subunit.

The expression of miR-124 increases in aged skin to cause cell senescence and it decreases in squamous cell carcinoma.

Skin senescence is induced by various factors including intrinsic aging and extrinsic aging. The current study compared the expression of microRNAs in young facial skin and senescent facial skin, and this study identified skin aging-related microRNAs. According to the results from a microRNA PCR Array, miR-124 was the microRNA that increased the most in senescent skin compared to young skin. Real-time PCR with a greater number of samples indicated that the increase in miR-124 levels in senescent facial skin was statistically significant. In situ hybridization was performed, and results indicated that the signal for miR-124 was evident in keratinocytes of senescent skin but not in those of young skin. The morphology of cultured normal human epidermal keratinocytes (NHEKs) transfected with a miR-124 mimic changed to an enlarged and irregular shape. In addition, the number of NHEKs positive for senescence-associated β-galactosidase (SA-β-gal) increased significantly as a result of the overexpression of the miR-124 mimic. The expression of miR-124 increased in UVB-irradiated NHEKs compared to controls in a dose-dependent manner. Expression of miR-124 in A431, a human cutaneous squamous cell carcinoma (SCC) cell line, decreased significantly compared to that in NHEKs. Forced overexpression of miR-124 as a result of the transfection of a miR-124 mimic in A431 resulted in the significant suppression of the proportion of cancer cells. The current results indicated that miR-124 increases as a result of cell senescence and that it decreases during tumorigenesis. The effect of supplementation of miR-124 in an SCC cell line suggests that senescence induction therapy with microRNA may be a new therapeutic approach for treatment of SCC.

Transforming growth factor β inhibitor Repsox down-regulates collagen expression of scleroderma dermal fibroblasts and prevents bleomycin-induced mice skin fibrosis

Inhibition of transforming growth factor (TGF)‐β1 signalling may be one of the most reliable approaches to treat skin fibrosis of scleroderma. Although there have been many basic researches of TGF‐β blockade reagents, few of them were proved to have inhibitory effects on fibrosis both in vitro and in vivo. In this study, we randomly chose four commercially available low molecular weight compounds (Repsox, LY2109761, LY364947 and K02288) from TGF‐β1 inhibitor library, and compared their antifibrotic effects in vitro and in vivo. We demonstrated that Repsox has the most potent inhibitory effects on TGF‐β‐induced expression of CTGF and collagen of cultured normal dermal fibroblasts in vitro and their constitutive overexpression of scleroderma fibroblast in vitro. In addition, Repsox could attenuate skin fibrosis by bleomycin in vivo, via the downregulation of CTGF or collagen. Our results may facilitate clinical trial of Repsox against fibrotic diseases in future.

Mice overexpressing integrin αv in fibroblasts exhibit dermal thinning of the skin

BACKGROUND Integrins, especially αv integrin (ITGAV), are thought to play central roles in tissue fibrosis and the pathogenesis of scleroderma. So far, skin phenotype of tissue-specific transgenic mice of ITGAV have not been investigated. OBJECTIVE To investigate the role of ITGAV in the skin fibrosis, we engineered transgenic mice that overexpress ITGAV in the fibroblasts under the control of the COL1A2 enhancer promoter. METHODS Protein or RNA expression was evaluated by real-time PCR, immunohistochemistry, immunoblotting and immunoprecipitation. RESULTS Dermal thickness and Massons trichrome staining were decreased in ITGAV transgenic (Tg) mice compared with wild-type (WT) mice. Protein and mRNA levels of COL1A2, COL3A1, CTGF and integrin β3 were down-regulated in the skin of Tg mice. In addition, the cell proliferation of cultured dermal fibroblasts obtained from Tg mice skin was decreased compared to those of WT mice. FAK phosphorylation was reduced in fibroblasts cultured from Tg mice skin in comparison to WT mice fibroblasts. Integrin β3 siRNA inhibited FAK phosphorylation levels, while FAK inhibitor reduced the expression of collagens and CTGF in mice dermal fibroblasts. CONCLUSIONS The down-regulation of collagen or CTGF by decreased integrin β3 and FAK phosphorylation may cause the dermal thinning in Tg mice. Lower CTGF may also result in reduced growth of Tg mice fibroblasts. Our hypothesis is that the balance between α and β chain of integrins positively or negatively control collagen expression and dermal thickness. This study gave a new insight in the treatment of tissue fibrosis and scleroderma by balancing integrin expression.

Expression of Let-7 family microRNAs in skin correlates negatively with severity of pulmonary hypertension in patients with systemic scleroderma

Background Pulmonary hypertension (PH) is a serious complication in patients with systemic scleroderma (SSc), therefore it is important to identify the factors that could predict the presence and progression of PH. Skin biopsy is performed in patients with SSc to examine the type and severity of the disease. MicroRNAs (miRNAs) are potential biomarkers for various cardiovascular diseases including PH. Methods and results We determined the skin miRNA expression profile in 15 SSc patients with (n = 6) and without PH (n = 9). A mixture of equal amounts of miRNAs from PH and non-PH patients were prepared and used for miRNA PCR array analysis. The analysis identified 591 upregulated miRNAs and 57 downregulated miRNAs in the PH group. Of these, only miRNAs with a Ct value of less than 35 were subjected to further analysis. When a 1.5-fold difference was considered meaningful, 32 miRNAs were upregulated and 14 miRNAs were downregulated in the PH group. Interestingly, 5 out of 14 downregulated miRNAs belonged to the let-7 family. The results were validated by quantitative real-time PCR with specific primer for each miRNA, which showed significant downregulation of five let-7 family members (let-7a, -7d, -7e, -7f, -7g) in 6 PH compared with 9 non-PH skin samples. The expression levels of let-7d and 7b correlated negatively with pulmonary arterial pressure measured by echocardiography. Conclusions The results suggest that skin miRNA is a potentially useful marker for the presence and severity of PH in patients with SSc.

microRNA level is raised in the hair shafts of patients with dematomyositis in comparison with normal subjects and patients with scleroderma.

The diagnosis of dermatomyositis is sometimes difficult. We tried to evaluate the possibility that levels of Homo sapiens microRNA‐214 (hsa‐miR‐214) in hair roots or hair shafts can be a useful marker of the disease.