Defining the role of miR-30 in human skin

描述(申请人提供)：太阳紫外线(UV)辐射是一种主要的环境皮肤致癌物质，可诱导DNA损伤，并调节调节细胞生长、增殖和凋亡的各种基因。基因的转录调控是细胞反应的一部分，细胞反应是抵御紫外线辐射不利影响的一种防御机制。MicroRNAs(MiRNA)是一组小的非编码RNA，通过靶向3‘非翻译区的序列来调节基因的功能。在包括皮肤癌在内的各种恶性肿瘤中，已经观察到miRNA表达的中断。关于miRNAs在人体皮肤对紫外线辐射反应的基因表达调控中的作用，人们知之甚少。我们选择了40mJ/cm2的UVB剂量，建立了UVB照射的正常人表皮角质形成细胞(NHEKs)的miRNA图谱。在我们的初步微阵列实验中，我们鉴定了44个miRNAs的子集，这些miRNAs在UVB暴露后4小时在NHEK中显著差异表达(p&lt；0.05)。这一数据进一步得到了qPCR的验证，发现总共有22个miRNAs受到UVB的调控。额外的统计测试显示，miR-30、miR-24和miR-222在两个实验系统中都有显著的调制。对暴露在UVB辐射下的三维表皮结构的研究证实了单层细胞培养的结果，并证明了miR-30的显著下调。我们的数据表明miR-30在UVB暴露的人体皮肤中具有潜在的作用。我们在这里测试的假设是，UVB介导的miR-30表达的减少导致了对与人类皮肤癌发病有关的增殖途径的抑制控制的丧失。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点预测位点来筛选miR-30的靶基因。将进行功能研究，以验证具有潜在作用的miR-30靶标 在紫外线诱导的反应中利用miRNA进行功能增减实验。这些结果将在一个多层的、高度分化的、接近模拟人类皮肤的三维人类表皮皮肤模型中得到验证。对于我们的体外数据的体内相关性，我们将检测人类皮肤样本中的miR-30及其靶基因，并研究miR-30表达水平与皮肤癌发生之间的可能相关性。我们的研究将深入了解miR-30在人体皮肤中的功能，并阐明其在紫外线诱导皮肤癌中的作用。

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.