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.

miR-150 down-regulation contributes to the constitutive type I collagen overexpression in scleroderma dermal fibroblasts via the induction of integrin ß3.

Overexpression of integrins in dermal fibroblasts is thought to play a key role in the pathogenesis of systemic sclerosis (SSc), but the mechanism is unknown. We evaluated the possibility that microRNAs (miRNAs) are involved in the regulation of integrin ß3 in these cells. The miRNA expression profile was determined by miRNA PCR array and real-time PCR. Protein expression of integrin ß3 was determined by immunoblotting. In vivo detection of miRNA in paraffin section was performed by in situ hybridization. miR-150 expression was decreased in SSc fibroblasts both in vivo and in vitro. The transfection of miR-150 inhibitor into normal fibroblasts induced expression of integrin ß3, phosphorylated Smad3, and type I collagen, whereas forced overexpression of the miRNA resulted in their down-regulation in SSc fibroblasts. Treatment of SSc fibroblasts with 5-AdC revealed that miR-150 down-regulation in these cells is caused by DNA methylation. In addition, we found that miR-150 is detectable and quantitative in serum. Serum miR-150 levels were decreased in SSc patients, and the SSc patients with lower serum miR-150 levels tended to have more severe clinical manifestations. miR-150 may play an important role in the pathogenesis of SSc via overexpression of integrin ß3. Investigation of the regulatory mechanisms of tissue fibrosis by miR-150 could lead to development of new diagnostic tools and new treatments using miRNA.

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.