基础医学与临床 ›› 2013, Vol. 33 ›› Issue (12): 1532-1537.

• 研究论文 • 上一篇    下一篇

CpG岛靶向siRNA诱导P16沉默的甲基化分析

杨晓红1,黄孝天1,朱伟锋2,刘卓琦2,余乐涵2,李华3,王晟1,罗达亚1   

  1. 1. 南昌大学基础医学院
    2. 南昌大学医学院
    3. 南昌大学医学实验教学部
  • 收稿日期:2012-11-09 修回日期:2013-03-31 出版日期:2013-12-05 发布日期:2013-11-28
  • 通讯作者: 罗达亚 E-mail:luodaya@hotmail.com
  • 基金资助:
    自发性高血压大鼠心、脑、肾和肠系膜微动脉缝隙连接特性的比较研究》国家自然科学基金(国家自然科学基金);江西省教育厅科技计划;南昌大学科研基金

DNA methylation analysis for CpG island targeted siRNA induced P16 silencing

  • Received:2012-11-09 Revised:2013-03-31 Online:2013-12-05 Published:2013-11-28
  • Contact: Da-Ya LUO E-mail:luodaya@hotmail.com

摘要: 目的 探讨RNA介导DNA甲基化(RdDM)是否参与哺乳动物细胞中P16基因的甲基化沉默。方法 将靶向P16基因CpG岛不同位点(启动子区和外显子区)的小干扰RNA(siRNA),分别转染人胃癌细胞株BGC823,用RT-PCR、免疫印迹检测P16基因的表达,用甲基化特异性PCR(MSP)和克隆测序等分析DNA甲基化。结果 靶向P16基因启动子与外显子区域的siRNA均能诱导P16基因表达的降低,但两者都不能诱导同源序列DNA甲基化。结论 在BGC823细胞株上,siRNA不能通过甲基化诱导方式调控P16基因的表达。

关键词: CpG岛, RNA介导DNA甲基化, siRNA, p16, 甲基化

Abstract: Objective P16 CpG island targeted short interfering RNAs (siRNAs) were designed to analyze whether RNA-directed DNA methylation (RdDM) functions in formation of P16 methylation in mammalian cells. Methods BGC823 cells were transduced with siRNAs targeted to promoter and exon1 region of P16, respectively. Semi-quantitative RT-PCR and western blot were used for P16 expression detection. DNA methylation statuses were also analyzed with methylation specific PCR (MSP) and bisulfite clone sequencing. Results The promoter and exon1 targeted siRNAs could decrease P16 expression. Nevertheless, both siRNAs could not induce methylation of targeted DNA. Conclusions The present data indicates that siRNAs can not down-regulate P16 gene expression through inducing DNA methylation in BGC823 cell line.

Key words: CpG island, RNA-directed DNA Methylation, siRNA, p16, methylation

中图分类号: