Defining the role of miR-30 in human skin

描述（由申请人提供）：太阳紫外线（UV）辐射是一种主要的环境致癌物，可诱导DNA损伤并调节多种调节细胞生长、增殖和凋亡的基因。基因的转录调控是细胞反应的一部分，作为防御紫外线辐射不利影响的机制。MicroRNAs （miRNA）是一组小的非编码rna，通过靶向其3'非翻译区序列来调节基因功能。在包括皮肤癌在内的各种恶性肿瘤中都观察到miRNA表达的破坏。我们对mirna在人体皮肤紫外线照射下基因表达调控中的作用知之甚少。我们生成了UVB照射下正常人表皮角质形成细胞（NHEKs）的miRNA谱，选择了生理相关的UVB剂量40mJ/cm2。在我们初步的微阵列实验中，我们发现了44个mirna的子集，在UVB暴露后4小时在NHEKs中显著（p< 0.05）表达差异。该数据通过qPCR进一步验证，共发现22个mirna被UVB调节。额外的统计检验显示，在两种实验系统中，miR-30、miR-24和miR-222都被显著调节。对暴露于UVB照射下的三维表皮结构的研究证实了单层细胞培养结果，并显示miR-30显著下调。我们的数据表明miR-30在UVB暴露的人类皮肤中具有潜在的作用。我们在这里验证的假设是，uvb介导的miR-30表达降低导致对与人类皮肤癌发病机制有关的增殖途径的抑制控制丧失。在确定miR-30参与UVB诱导的皮肤反应后，我们将利用mRNA微阵列平台和计算靶标预测位点来筛选miR-30的靶基因。我们将进行功能研究来验证miR-30靶点的潜在作用

DESCRIPTION (provided by applicant): Solar ultraviolet (UV) radiation is a major environmental skin carcinogen that induces DNA damage and modulates a variety of genes that regulate cell growth, proliferation and apoptosis. The transcriptional regulation of genes is part of the cellular reaction that operates as a defense mechanism against the adverse effects of UV radiation. MicroRNAs (miRNA) are a group of small non-coding RNAs which regulate gene functions by targeting sequences in their 3' untranslated regions. Disruption of miRNA expression has been observed in various malignancies including skin cancers. Very little is known about the role of the miRNAs in the regulation of gene expression in response to UV irradiation in human skin. We generated a miRNA profile of UVB-irradiated normal human epidermal keratinocytes (NHEKs) selecting the physiologically relevant UVB dose of 40mJ/cm2. In our preliminary microarray experiments, we identified a subset of 44 miRNAs that were significantly (p< 0.05) differentially expressed in NHEKs, 4 h post UVB exposure. This data was further validated by qPCR which revealed a total of 22 miRNAs that were modulated by UVB. Additional statistical testing showed miR-30, miR-24 and miR-222 to be significantly modulated in both experimental systems. Studies in 3-D epidermal constructs exposed to UVB irradiation confirmed monolayer cell culture findings and demonstrated significant downregulation of miR-30. Our data indicate a potential role of miR-30 in the UVB exposed human skin. The hypothesis we test here is that UVB-mediated decrease in miR-30 expression results in loss of inhibitory control of proliferative pathways implicated in the pathogenesis of human skin cancer. After establishing the involvement of miR-30 in UVB induced skin response, we will shortlist the target genes of miR-30, employing mRNA microarray platform and computational target prediction sites. Functional studies will be conducted to verify miR-30 targets with potential role in UV-induced response using miRNA gain- and loss-of-function experiments. These results will be validated in a multilayered, highly differentiated, 3-D human epidermal skin model closely simulating human skin. For in vivo relevance of our in vitro data, we will examine miR-30 and its target genes in human skin specimens and study a possible correlation between miR-30 expression levels and the occurrence of skin cancer. Our study will provide in-depth understanding of the functions of miR-30 in the human skin and delineate its role in UV- induced skin cancer.