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皮肤致癌物，可诱导DNA损伤并调节调节细胞生长、增殖和凋亡的多种基因。基因的转录调控是细胞反应的一部分，作为抵御紫外线辐射不利影响的防御机制。MicroRNA（miRNA）是一类非编码小RNA，通过靶向3'端非翻译区序列调控基因功能。在包括皮肤癌在内的各种恶性肿瘤中已经观察到miRNA表达的破坏。关于miRNAs在人类皮肤中响应于紫外线照射的基因表达调节中的作用知之甚少。我们产生了UVB照射的正常人表皮角质形成细胞（NHEK）的miRNA谱，选择生理相关的UVB剂量为40 mJ/cm 2。在我们初步的微阵列实验中，我们鉴定了UVB暴露后4小时在NHEK中显著（p< 0.05）差异表达的44种miRNA的子集。通过qPCR进一步验证了该数据，qPCR揭示了受UVB调节的总共22种miRNA。额外的统计学检验显示miR-30、miR-24和miR-222在两个实验系统中均被显著调节。暴露于UVB照射的3-D表皮构建体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暴露的人类皮肤中的潜在作用。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点预测位点来筛选miR-30的靶基因。将进行功能研究以验证miR-30靶标的潜在作用 在紫外线诱导的反应中使用miRNA功能获得和丧失实验。这些结果将在一个多层的，高度分化的，3-D人体表皮皮肤模型密切模拟人体皮肤进行验证。对于我们的体外数据的体内相关性，我们将检查人类皮肤标本中的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。我们的研究将提供对miR-30在人类皮肤中的功能的深入理解，并描述其在UV诱导的皮肤癌中的作用。

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.