MiRNA与转录后调控.pptVIP

应用芯片技术高通量的检测不同样品中多种miRNA的表达量的差异 NAR 33(2)，2005  * NAR 33(2)，2005  * Science 309(5732):310-311,2005. 原位杂交技术可以方便的检测miRNA的时空表达的差异 神经系统 肝脏 肌肉 血管和心脏 侧线和感官系统 前肾 * 3.miRNA的功能的研究方法 ○ 高表达miRNA（转基因） ○ 在体内抑制miRNAs的表达 * PLoS BIOLOGY 2(4):E98, 2004 通过对miRNA的合成及功能行使过程所必需的蛋白复合物进行抑制从而降低miRNA的表达 在体内使miRNA的表达量下调 2‘-O-甲基修饰的与let7互补的寡核苷酸的导入可以使let7丧失功能，是由于这种带修饰的寡核苷酸的结合封闭了RISK复合体 * Nature 423, 2003 针对miRNA的前体的发夹结构设计siRNA降低miRNA的表达量 * Curr.Opin.Chem.Biol. 7(4):516-523,2003. 1. 染色体重塑 2. DNA甲基化 3. 转录 4. RNA 加工 5. RNA 稳定性 6. RNA 转运 7. RNA定位 8. 翻译 9. 蛋白质活性 10. miRNA 稳定性 miRNAs可能参与各种基因表达过程的调控 * (V) miRNA and SNPs * Functional analysis of SNPs Promoter Intron Exon2 Exon1 5’UTR 3’UTR Functions of different elements above: Promoter: Transcription 5’UTR: Transcription and translation Coding region: Function and activity of enzymes; Translation efficiency Intron: Alternative splicing 3’UTR: mRNA Stability, mRNA location and translation * Association study of MDM2 234 bladder cancer cases, 253 controls. 5 out of 18 tagSNPs are positive (P0.05) . One fascinating positive SNP in the 3’UTR 460TC (P=0.01). mRNA: 5’UTR Coding region 3’UTR * microRNA target prediction microRNA prediction: Based on “ seed sequence” Pic Tar （) Targetscan （) * SNP TC Putative microRNAs Has-mir-25/32/92/323/367 Targetscan （) * SNP modulate miR-25 binding on structure and stability Predicted by Mfold * SNP modulate miR-32 binding on structure and stability * There are also predicted microRNAs, which are not likely to be real regulators. Taking miR-92 for Example: Inadequate basepairing Low thermodynamic stability * Luciferase Assay to Confirm Our Predictions Luciferase REV3L 3’UTR Poly A Luciferase REV3L 3’UTR Poly A Luciferase REV3L Poly A T Allele C Allele 3’UTR 11bp Deletion of putative microRNA target 1 2 3 * Bladder derived T24 cell: * *p0.05 *** P0.0001 11bp Deletion: Deletion of putative microRNA targets *