Shinichi Masuguchi

The circulating microRNA-221 level in patients with malignant melanoma as a new tumor marker

BACKGROUND MicroRNA-221 (miR-221) is known to be abnormally expressed in malignant melanoma (MM) cells, and it favors the induction of the malignant phenotype through down-modulation of p27Kip1/CDKN1B and the c-KIT receptor. This suggests that the serum level of miR-221 might increase in patients with MM and thus could be used as a new tumor marker. OBJECTIVE To evaluate the possibility that the serum miR-221 level can be a marker of MM. METHODS Serum samples were obtained from 94 MM patients and 20 healthy controls. MicroRNAs were purified from serum, and miR-221 levels were measured by quantitative real-time polymerase chain reaction. RESULTS Circulating miR-221 was detectable and could be quantified in serum samples. MM patients had significantly higher miR-221 levels than healthy controls. Among the MM patients, the miR-221 levels were significantly increased in patients with stage I-IV MM compared to those with MM in situ, and the levels were correlated with tumor thickness. Moreover, a longitudinal study revealed a tendency for the miR-221 levels to decrease after surgical removal of the primary tumor, and to increase again at recurrence. CONCLUSIONS Serum levels of miR-221 were significantly increased in MM patients and may be useful not only for the diagnosis of MM, but also for the differentiating MM in situ from stage I-IV MM, and for evaluating tumor progression and monitoring patients during the follow-up period. In addition, considering that the serum levels of miR-221 were correlated with tumor thickness, miR-221 might also be useful as a prognostic marker for patients with MM.

Impaired IL-17 Signaling Pathway Contributes to the Increased Collagen Expression in Scleroderma Fibroblasts

Among IL-17 families, IL-17A and IL-17F share amino acid sequence similarity and bind to IL-17R type A. IL-17 signaling is implicated in the pathogenesis of various autoimmune diseases, but its role in the regulatory mechanism of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) both remain to be elucidated. This study revealed that IL-17A expression was significantly increased in the involved skin and sera of SSc patients, whereas the IL-17F levels did not increase. In contrast, the expression of IL-17R type A in SSc fibroblasts significantly decreased in comparison with that in normal fibroblasts, due to the intrinsic TGF-β1 activation in these cell types. Moreover, IL-17A, not IL-17F, reduced the protein expression of α1(I) collagen and connective tissue growth factor. miR-129-5p, one of the downregulated microRNAs in SSc fibroblasts, increased due to IL-17A and mediated the α1(I) collagen reduction. These results suggest that IL-17A signaling, not IL-17F, has an antifibrogenic effect via the upregulation of miR-129-5p and the downregulation of connective tissue growth factor and α1(I) collagen. IL-17A signaling is suppressed due to the downregulation of the receptor by the intrinsic activation of TGF-β1 in SSc fibroblasts, which may amplify the increased collagen accumulation and fibrosis characteristic of SSc. Increased IL-17A levels in the sera and involved skin of SSc may be due to negative feedback. Clarifying the novel regulatory mechanisms of fibrosis by the cytokine network consisting of TGF-β and IL-17A may lead to a new therapeutic approach for this disease.

TGF-β–Mediated Downregulation of MicroRNA-196a Contributes to the Constitutive Upregulated Type I Collagen Expression in Scleroderma Dermal Fibroblasts

Previous reports indicated the significance of the TGF-β signaling in the pathogenesis of systemic sclerosis. We tried to evaluate the possibility that microRNAs (miRNAs) play a part in the type I collagen upregulation seen in normal fibroblasts stimulated with exogenous TGF-β and systemic sclerosis (SSc) fibroblasts. miRNA expression profile was evaluated by miRNA PCR array and real-time PCR. The protein expression of type I collagen was determined by immunoblotting. In vivo detection of miRNA in paraffin section was performed by in situ hybridization. Several miRNAs were found to be downregulated in both TGF-β–stimulated normal fibroblasts and SSc fibroblasts compared with normal fibroblasts by PCR array. Among them, miR-196a expression was decreased in SSc both in vivo and in vitro by real-time PCR or in situ hybridization. In SSc fibroblasts, miR-196a expression was normalized by TGF-β small interfering RNA. miR-196a inhibitor leads to the overexpression of type I collagen in normal fibroblasts, whereas overexpression of the miRNA resulted in the downregulation of type I collagen in SSc fibroblasts. In addition, miR-196a was detectable and quantitative in the serum of SSc patients. Patients with lower serum miR-196a levels had significantly higher ratio of diffuse cutaneous SSc:limited cutaneous SSc, higher modified Rodnan total skin thickness score, and higher prevalence of pitting scars than those without. miR-196a may play some roles in the pathogenesis of SSc. Investigation of the regulatory mechanisms of type I collagen expression by miR-196a may lead to new treatments using miRNA.

The Downregulation of microRNA let-7a Contributes to the Excessive Expression of Type I Collagen in Systemic and Localized Scleroderma

Systemic and localized scleroderma (SSc and LSc) is characterized by excessive deposition of collagen and tissue fibrosis in the skin. Although they have fundamental common characteristics including autoimmunity, little is known about the exact mechanism that mediates the excessive collagen expression in these disorders. In the current study, we tried to evaluate the possibility that microRNAs (miRNAs) play some roles in the pathogenesis of fibrosis seen in these diseases. miRNA expression patterns were evaluated by miRNA array analysis, real-time PCR, and in situ hybridization. The function of miRNAs in dermal fibroblasts was assessed using miRNA inhibitors, precursors, or protectors. In the mouse model of bleomycin-induced dermal sclerosis, the overexpression of miRNAs was performed by i.p. miRNA injection. We demonstrated let-7a expression was downregulated in SSc and LSc skin both in vivo and in vitro, compared with normal or keloid skin. The inhibition or overexpression of let-7a in human or mouse skin fibroblasts affected the protein expression of type I collagen or luciferase activity of collagen 3′-untranslated region. Also, we found let-7a was detectable and quantitative in the serum and investigated serum let-7a levels in patients with SSc or LSc. let-7a concentration was significantly decreased in these patients, especially in LSc patients. Moreover, we revealed that the intermittent overexpression of let-7a in the skin by i.p. miRNA injection improved the skin fibrosis induced by bleomycin in mice. Investigation of more detailed mechanisms of miRNA-mediated regulation of collagen expression may lead to new therapeutic approaches against SSc and LSc.

Down-regulation of mir-424 contributes to the abnormal angiogenesis via MEK1 and cyclin E1 in senile hemangioma: Its implications to therapy

Background Senile hemangioma, so-called cherry angioma, is known as the most common vascular anomalies specifically seen in the aged skin. The pathogenesis of its abnormal angiogenesis is still unclear. Methodology/Principal Findings In this study, we found that senile hemangioma consisted of clusters of proliferated small vascular channels in upper dermis, indicating that this tumor is categorized as a vascular tumor. We then investigated the mechanism of endothelial proliferation in senile hemangioma, focusing on microRNA (miRNA). miRNA PCR array analysis revealed the mir-424 level in senile hemangioma was lower than in other vascular anomalies. Protein expression of MEK1 and cyclin E1, the predicted target genes of mir-424, was increased in senile hemangioma compared to normal skin or other anomalies, but their mRNA levels were not. The inhibition of mir-424 in normal human dermal microvascular ECs (HDMECs) using specific inhibitor in vitro resulted in the increase of protein expression of MEK1 or cyclin E1, while mRNA levels were not affected by the inhibitor. Specific inhibitor of mir-424 also induced the cell proliferation of HDMECs significantly, while the cell number was decreased by the transfection of siRNA for MEK1 or cyclin E1. Conclusions/Significance Taken together, decreased mir-424 expression and increased levels of MEK1 or cyclin E1 in senile hemangioma may cause abnormal cell proliferation in the tumor. Senile hemangioma may be the good model for cutaneous angiogenesis. Investigation of senile hemangioma and the regulatory mechanisms of angiogenesis by miRNA in the aged skin may lead to new treatments using miRNA by the transfection into senile hemangioma.

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.

microRNA-92a expression in the sera and dermal fibroblasts increases in patients with scleroderma

OBJECTIVES microRNAs (miRNAs) play a part in various cellular activities. However, the role of miRNA in SSc is not fully understood. This study investigated the expression and role of miR-92a in SSc patients and evaluated the possibility that miR-92a is involved in the pathogenesis of this disease. METHODS Serum samples were obtained from 61 SSc patients. mRNAs were purified from serum and levels of miR-92a and miR-135 were measured with quantitative real-time PCR. miR-92a expression in dermal fibroblasts was also determined by quantitative real-time PCR. Immunoblotting was performed to detect MMP-1 protein. RESULTS The median serum levels of miR-92a, not miR-135, were significantly higher in SSc patients than normal subjects. The constitutive up-regulated miR-92a expression was also found in cultured dermal fibroblasts from SSc skin, which was decreased by the transfection with siRNA of TGF-β. Furthermore, the forced overexpression of miR-92a in normal dermal fibroblasts using miR-92a mimic resulted in the down-regulation of MMP-1 expression. CONCLUSION The increase of miR-92a in SSc may be due to the stimulation of intrinsic TGF-β activation seen in this disease. There is also a possibility that MMP-1 is the target of miR-92a and that increased miR-92a expression therefore plays a role in excessive collagen accumulation in SSc via the down-regulation of MMP-1. Clarifying the role of miRNAs in SSc may result in a better understanding of this disease and the development of new therapeutic approaches.

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.

Down-regulation of miR-124/-214 in cutaneous squamous cell carcinoma mediates abnormal cell proliferation via the induction of ERK.

Squamous cell carcinoma (SCC) is one of the most common skin cancers. Because its potential to recur and metastasize leads to a poor prognosis and significant mortality, it is necessary to develop new early diagnostic tools and new therapeutic approaches. In this study, we found protein levels of ERK1 and ERK2 were increased in SCC cell lines without changing mRNA levels and that ERK1/2 mediates abnormal cell proliferation in these cells. Then, mechanisms underlying the overexpression of ERK1/2 in SCC were investigated focusing on microRNA. We found that miR-214 is the regulator of ERK1, whereas ERK2 is regulated by miR-124 and miR-214. Expression of miR-124 and miR-214 was significantly down-regulated in SCC in vitro and in vivo. Treatment with 5-aza-deoxycytidine and trichostatin A synergistically recovered the miR-124/-214 down-regulation in SCC cell line. However, bisulphite sequencing revealed the methylation status of miR-124/-214 promoter was not increased in the SCC cell line and tumor tissue. Taken together, the down-regulation of miR-124/-214 in SCC is most likely caused, at least in part, by hypermethylation of other promoter regions rather than the miR-124/-214 promoter. Supplementation of these microRNAs in the SCC cell line reduced the abnormal cell proliferation by normalizing ERK1/2 levels. Additionally, serum concentration of miR-124 was correlated with miR-124 expression levels in the tumor tissues and inversely correlated with tumor progression. On the other hand, miR-214 was not detected in the serum. Investigation of the regulatory mechanisms of keratinocyte proliferation by microRNA may lead to develop new biomarkers and treatments using microRNA.

Increased accumulation of extracellular thrombospondin-2 due to low degradation activity stimulates type i collagen expression in scleroderma fibroblasts

The aim of the present study was to determine the expression and role of thrombospondin-2 (TSP-2) in systemic sclerosis (SSc). Both TSP-2 mRNA levels and protein synthesis in cell lysates were significantly lower in cultured SSc fibroblasts than in normal fibroblasts; however, the TSP-2 protein that accumulated in the conditioned medium of SSc fibroblasts was up-regulated, compared with that of normal fibroblasts, because of an increase in the half-life of the protein. In vivo serum TSP-2 levels were higher in SSc patients than in healthy control subjects, and SSc patients with elevated serum TSP-2 levels tended to have pitting scars and/or ulcers. TSP-2 knockdown resulted in the down-regulation of type I collagen expression and the up-regulation of miR-7, one of the miRNAs with an inhibitory effect on collagen expression. Expression levels of miR-7 were also up-regulated in SSc dermal fibroblasts both in vivo and in vitro. Taken together, these findings suggest that the increased extracellular TSP-2 deposition in SSc fibroblasts may contribute to tissue fibrosis by inducing collagen expression. Down-regulation of intracellular TSP-2 synthesis and the subsequent miR-7 up-regulation in SSc fibroblasts may be due to a negative feedback mechanism that prevents increased extracellular TSP-2 deposition and/or tissue fibrosis. Thus, TSP-2 may play an important role in the maintenance of fibrosis and angiopathy in patients with SSc.