新疆维吾尔族白癜风microRNA的筛选及调控机制研究

Vitiligo is a common, acquired and chronic pigmentary disorder of discosmetic dermatosis. Our earlier studies have shown the distinctive clinical characteristics of Uygur patients with vitiligo. In different nation and race, the incidence of vitiligo has genetic heterogeneity. In addition, 10 differentially expressed miRNA in Uygur vitiligo has been screened out through miRNA microarray in our preliminary experiment. Therefore, we intend to use miRNA microarray to detect the differentially expressed miRNA. Comparing the results with preliminary experiment, and screening 3 largest differentially expressed miRNA to validate the microarray results in plasma and fresh tissues simultaneously. The differences between the differentially expressed miRNA and clinical phenotype of vitiligo will be analyzed. Then the target gene of the differentially expressed miRNA will be predicted via TargetScan, etc. software. GO and KEGG are used to analyze the function and metabolic pathway of target genes. Synthetizing of mimics and inhibitors of differential miRNA and transfecting them into melanocytes, MTT and flow cytometry will be used to detect the cell proliferation, cell cycle and apoptosis. The changes of protein and mRNA expression levels before and after the transfection will be detected by Western Blot and qRT-PCR, respectively. To explain the regulatory role of differential miRNA in vitiligo, and to provide a new theoretical basis for the molecular mechanism of vitiligo.

白癜风是一常见的获得性、慢性色素脱失性疾病。前期研究发现维吾尔族白癜风有其独特的临床特点，且不同民族、种族白癜风发病具有遗传异质性。预实验通过miRNA芯片已筛选出了10条差异表达的miRNA。本项目拟采用miRNA芯片进一步筛选差异表达的miRNA，并与预实验芯片结果进行比对，筛选出差异性最大的3条miRNA在血浆和新鲜组织中同时验证，分析验证后仍差异表达的miRNA(差异miRNA)与维吾尔族白癜风临床特征的相关性。采用TargetScan等得到差异miRNA调控的靶基因，GO和KEGG对相关靶基因功能和代谢通路行富集分析。进一步合成差异miRNA的类似物和抑制物并转染至黑素细胞，通过MTT、流式检测细胞增殖、细胞周期和凋亡；采用qRT-PCR、Western Blot检测转染前后靶基因mRNA及蛋白的变化，阐释miRNA在白癜风中的调控作用，为白癜风的发生机制提供新的理论基础和依据。

白癜风是一常见的获得性、慢性色素脱失性疾病。不同民族白癜风发病具有遗传异质性。本项研究对乌鲁木齐市及周边地区白癜风患者临床特征进行分析，筛选维吾尔族白癜风血浆中差异表达的miRNA，验证差异表达miRNA；预测差异表达miRNA的靶基因，分析差异表达miRNA与靶基因的表达关系；分离并培养原代黑素细胞，通过细胞水平的功能学实验探究过表达或抑制差异表达miRNA后对细胞的生物学功能影响及对其靶基因在mRNA和蛋白水平表达的影响，采用双荧光素酶报告基因实验分析差异表达miRNA和其靶基因的调控机制。分析收集的516名白癜风患者临床特征，不同民族发病年龄有明显差异，其中维吾尔患者发病年龄明显早于汉族。选取15例维吾尔族白癜风患者及15例正常对照，通过miRNA芯片检测血浆中miRNA的表达情况，采用免疫致病机理相关的miRNA PCR Array检测血浆中miRNA 的表达水平，两者共同筛选到miR-223-3p，经过qRT-PCR检测验证，miR-223-3p在白癜风血浆中表达明显升高。基于TargetScan在线预测网站，对miR-223-3p的靶基因进行预测与筛选，基于生物信息学技术，初步筛选到FOXO3，经在白癜风公共数据集GSE75819、GSE53146、GSE90880中检测FOXO3的表达水平均为下调趋势。分离培养原代黑素细胞，合成差异表达miRNA的类似物和抑制物，转染到原代黑素细胞中，细胞水平功能学实验显示：在原代培养的黑素细胞中过表达miR-223-3p后，明显抑制了黑素细胞的增殖能力，促进了细胞的凋亡；而抑制miR-223-3p的表达后则表现为与过表达相反的效应。同时，过表达miR-223-3p后FOXO3在mRNA和蛋白水平表达均明显降低。双荧光素酶报告基因实验结果表明，miR-223-3p通过与FOXO3基因的3’UTR区直接结合而参与调控原代黑素细胞的增殖与凋亡。在白癜风血浆中miR-223-3p特异性高表达，miR-223-3p靶向作用于FOXO3，与FOXO3基因的3’UTR直接结合，miR-223-3p负向调控FOXO3表达，参与调控原代黑素细胞的增殖与凋亡，有望为白癜风的治疗靶点及研究方向提供新的选择。