中华皮肤科杂志 ›› 2013, Vol. 46 ›› Issue (4): 239-243.

• 论著 • 上一篇    下一篇

p53基因沉默对HaCaT细胞MicroRNA表达谱的影响

任金平1,王平2,洪为松3,韩飞4,黎钊5,倪亚杰6   

  1. 1. 浙江中医药大学附属杭州市第三人民医院
    2. 杭州市第三人民医院
    3. 杭州市第三人民医院皮肤科
    4. 安徽医科大学杭州临床医学院
    5. 浙江中医药大学附属杭州第三医院
    6. 浙江中医药大学
  • 收稿日期:2012-05-25 修回日期:2012-12-13 出版日期:2013-04-15 发布日期:2013-04-01
  • 通讯作者: 王平 E-mail:dermwang@aliyun.com
  • 基金资助:
    浙江省自然科学基金项目;杭州市医学重点专科专病项目

Effect of p53 gene silencing on microRNA expression profiles in HaCaT human keratinocytes

  • Received:2012-05-25 Revised:2012-12-13 Online:2013-04-15 Published:2013-04-01
  • Contact: Ping E-mail:dermwang@aliyun.com

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摘要： 目的 研究p53基因沉默前后HaCaT细胞microRNA（miRNA）的差异表达谱并进行相关功能分析。方法 利用慢病毒介导的RNAi对培养的HaCaT细胞株进行p53基因沉默，通过Trizol法抽提细胞总RNA，PEG（聚乙二醇）方法分离miRNA，T4RNA连接酶荧光标记后进行miRNA芯片杂交，利用Genepix 4000B 图像分析软件和Genepix Pro 6.0软件进行数据分析，生物信息学方法检索出p53基因沉默前后HaCaT细胞差异表达的miRNA调控的靶基因，选取每个miRNA调控的前20个靶基因进行靶基因功能及KEGG分析。结果 p53基因沉默前后HaCaT细胞中发现53个差异表达的miRNA，其中41个表达上调，12个下调（差异 ＞ 2倍）。上调超过200倍的miRNA有：hsa-miR-141-3p、hsa-miR-15a-5p、hsa-miR-27a-3p、hsa-miR-130b-3p、hsa-miR-19a-3p；下调超过75%的miRNA有：hiv1-miR-TAR-3p、hsa-miR-630、hsa-miR-1246、hsa-miR-1275。靶基因预测和靶基因KEGG分析结果显示，部分靶基因与MAPK信号通路、代谢通路、肿瘤侵袭等有关。结论 hsa-miR-141-3p等9个miRNA及其调控的靶基因可能参与p53的分子调控。 【关键词】 基因，p53； 角蛋白细胞； 微RNAs； 基因沉默； 芯片分析技术

关键词: 角蛋白细胞, 基因，p53, 微RNAs, 基因沉默, 芯片分析技术

Abstract: REN Jin-ping *, WANG Ping, HONG Wei-song, HAN Fei, LI Zhao, NI Ya-jie. * Department of Dermatology, Third People′s Hospital of Hangzhou Affiliated to Zhejiang University of Traditional Chinese Medicine, Hangzhou 310053, China Corresponding author: WANG Ping, Email: dermwang@yahoo.com.cn 【Abstract】 Objective To assess differential expression profiles of microRNAs（miRNAs） in HaCaT human keratinocytes before and after p53 gene silencing, and to make a functional analysis of target genes. Methods Lentivirus-mediated RNA interference （RNAi） was used to silence p53 gene in HaCaT cells. Total RNA was extracted using Trizol reagent. Then, miRNAs were isolated by polyethylene glycol （PEG） and subjected to fluorescent labeling using T4RNA ligase followed by hybridization to a mammalian miRNA chip. Microarrays were scanned by a GenePix 4000B microarray scanner and fluorescence ratios were determined with the GenePix Pro 6.0 software. The TargetScan software was used to predict target genes of differentially expressed miRNAs （>2-fold difference in expression level）, and the top 20 target genes with the highest enrichment score were selected for each miRNA and subjected to functional analysis and pathway analysis through the KEGG signaling database. Results Totally, 53 differentially expressed miRNAs, including 12 down-regulated and 41 up-regulated miRNAs, were identified in HaCaT cells after p53 silencing as compared to those before p53 silencing. Of these 53 differentially expressed miRNAs, 5 （hsa-miR-141-3p，hsa-miR-15a-5p，hsa-miR-27a-3p，hsa-miR-130b-3p，hsa-miR-19a-3p） showed a more than 200-fold increase in expression, and 4 （hiv1-miR-TAR-3p，hsa-miR-630，hsa-miR-1246，hsa-miR-1275） experienced a more than 4-fold decrease in expression in HaCaT cells after p53 silencing. Functional analysis and pathway analysis revealed that some target genes of these differentially expressed miRNAs were involved in the mitogen-activated protein kinase （MAPK） signaling pathway, metabolic pathways, and tumor invasion. Conclusion Nine miRNAs, including hsa-miR-141-3p, may be involved in p53-mediated molecular regulation. 【Key words】 Genes, p53; Keratinocytes; MicroRNAs; Gene silencing; Microchip analytical procedures